Compositions in the form of an injectable aqueous solution comprising human glucagon and a statistical co-polyamino acid

ABSTRACT

Physically stable compositions in the form of an injectable aqueous solution, the pH of which is from 6.0 to 8.0, includes at least: a) human glucagon, and b) a co-polyamino acid bearing carboxylate charges and hydrophobic radicals Hy. In an embodiment, the compositions include, in addition, a gastrointestinal hormone.

Human glucagon is a short-acting hyperglycemic hormone which makes itimpossible to increase glycemia, thus correcting a hypoglycemic levelthat can result from an insulin excess. It enables the release ofglucose by stimulation of hepatic glycogenolysis and it has insulinantagonistic properties (hypoglycemic). Human glucagon is normallysecreted by the alpha cells of the islets of Langerhans in the pancreaswhen hypoglycemia is detected.

Human glucagon is used for therapeutic purposes, such as the emergencytreatment of severe hypoglycemia, also referred to as “rescue,” but alsoin a diagnostic context in the performance of medical examinations, forexample, in order to inhibit gastrointestinal motility. Otherapplications are also considered for human glucagon, in particular itsuse in a bi-hormonal glycemia regulation system, also referred to asartificial pancreas, and in congenital hyperinsulinism, which is a raredisease characterized by very high insulin levels.

The clinical use of human glucagon has been limited because of some ofits disadvantageous properties in terms of development of a stablepharmaceutical product with therapeutic intent. Indeed, human glucagonhas a very low solubility at physiological pH and a high physicalinstability, due to its propensity to form fibrils in a large pH range.This is the reason why the only commercial products based on humanglucagon (Glucagen®, NOVO NORDISK and Glucagon for injection, ELI LILLY)are lyophilized forms to be reconstituted extemporaneously.

The studies of Onoue t al. (Pharm. Res. 2004, 21(7), 1274-83) have shownthe potentially dangerous characters of these fibrils: fibrillated humanglucagon being cytotoxic in mammalian cells in culture.

In addition to its physical instability, human glucagon undergoesvarious types of chemical degradation. In an aqueous solution, itdegrades rapidly to form several degradation products. At least 16degradation products of human glucagon have been identified by Kirsh etal. (International Journal of Pharmaceutics, 2000, 203, 115-125). Thechemical degradation of this human glucagon is thus rapid and complex.

The poor chemical and physical stability of human glucagon in solutionhas led pharmaceutical companies such as NOVO NORDISK, ELI LILLY andmore recently FRESENIUS KABI to market this human glucagon in the formof a lyophilizate to be reconstituted at acidic pH (pH<3) immediatelybefore injection. Human glucagon in lyophilizate form is more stable,and the preparation of the formulation at acidic pH immediately beforeuse makes it possible to obtain a clear solution. However, once theproduct is reconstituted, it has to be used rapidly, because itundergoes an extremely rapid chemical and physical degradation in theacidic reconstitution buffer, with appearance of human glucagon fibrilswithin 24 hours after the reconstitution and/or gelling of thecomposition. However, this presentation of the product is unsatisfactorybecause it requires a very rapid use of the formulation. Thisinstability not only makes it impossible to use it in a pump, but italso presents the disadvantage of leading to large product losses indiagnostic use. In fact, since a composition of this type is no longerusable a few hours after its preparation, this leads to wastage.

Finally, even in the application of an emergency treatment for severehypoglycemic reactions that can occur during insulin therapy in diabeticpatients, the formulation to be reconstituted is also not ideal becauseit involves a long and complicated preparation, for example, the packageinsert of GlucaGen® describes a 5-step procedure for injecting therecommended dose. Moreover, a study of the company LOCEMIA demonstratesthat very few persons (approximately 10% of the participants) who weresupposed to perform the reconstitution in emergency were capable ofdelivering the appropriate dose. Finally, the acidic pH of the solutionsof human glucagon can generate pain upon injection in the patient.

Thus, there is a need for a ready-to-use human glucagon solution. Today,the solutions that are most advanced clinically speaking in order toenable the delivery of human glucagon circumvent the problem ofstability of human glucagon in aqueous solution in different ways.

The company LOCEMIA has developed a lyophilized human glucagon spraywhich is currently being tested in phase 3 clinical study, and which isintended to be administered by the intranasal route. This spray issuitable for a so-called “rescue” use, that is to say in the case of asevere hypoglycemia, since it is ready to use and thus easy to use, incontrast to the solutions that need to be reconstituted. However, thisproduct is not suitable for use in a pump or for a use that requires aprecise control of the delivered quantity of human glucagon.

As for XERIS, it has developed a liquid formulation of human glucagonbased on a polar aprotic solvent, such as DMSO, which is currently beingtested in clinical studies. However, while the injection of a solutionof organic solvents for “rescue” use can be considered, it is largelypreferable to have an aqueous human glucagon solution for chronic use.Compositions comprising a combination with other peptides have beenconsidered, notably amylin or a GLP-1 RA (glucagon-like peptide-1receptor agonist).

Finally, in the face of the difficulties of formulation of humanglucagon, analogs of human glucagon are in the process of beingdeveloped by large pharmaceutical companies such as NOVO NORDISK, SANOFIor ELI LILLY, in order to obtain formulations that have a stabilitycompatible with pharmaceutical use. However, these peptides of which theprimary sequence has been modified in comparison to the peptide of humanorigin can present a safety risk for the patients.

Thus, there is a major advantage in a solution that makes it possible toimprove the solubilization and the stability, both chemical andphysical, of human glucagon in an aqueous solution at a pH close tophysiological pH, that is to say from 6.0 to 8.0. This could make itpossible to obtain a pharmaceutical product that is easier to use by thepatient in an emergency, but it could also open the field to newtherapeutic applications of human glucagon, such as its use in abi-hormonal artificial pancreas, for example.

The prior art proposes solutions in order to attempt to solve thisproblem.

Some documents propose using an alkaline pH. For example, US2015291680teaches the solubilization of human glucagon at 1 mg/mL by using a pHfrom 8.8 to 9.4 and by using ferulic acid or tetrahydrocurcumin.However, apart from the fact that an alkaline pH is used, this solutionpresents the disadvantage of leading to a rather limited stability ofthe human glucagon over time. The article by Jackson et al. (Curr. Diab.Rep., 2012, 12, 705-710) proposes formulating human glucagon at analkaline pH (approximately 10), in order to limit the formation offibrils. However, this solution does not prevent a rapid chemicaldegradation of human glucagon.

The application WO2014096440 (NOVOZYME), on the other hand, considersusing a slightly acidic pH (approximately 5.5) in the presence ofalbumin and polysorbate, in order to improve the stability by reducingthe rate of fibril formation. However, this solution presents a limitedimprovement of the stability. Most of the solutions described in theprior art making it possible to obtain a clear solution of humanglucagon and to prevent aggregation, gelling or precipitation of thehuman glucagon involve the use of surfactants, detergents or other knownsolubilizing agents.

For example, Matilainen et al. (J. Pharm. Sci., 2008, 97, 2720-2729 andEur. J. Pharm. Sci., 2009, 36, 412-420) described the use ofcyclodextrin in order to limit the rate of formation of fibrils of humanglucagon. However, the improvement provided does not appear to besufficient for considering use in a pump.

The solutions proposed comprise hydrophilic surfactants:

-   -   GB1202607 (NOVO NORDISK) describes the use of anionic or        cationic detergents.    -   U.S. Pat. No. 6,384,016 (NOVO NORDISK) and US2011097386 (BIODEL)        use lysophospholipids (or lysolecithins).    -   WO2015095389 (AEGIS) describes non-ionic surfactants such as        dodecyl maltoside for improving the bioavailability of        therapeutic agents, in the case of delivery by application on        the mucous membranes or the epidermis, and, in particular, in        the case of ocular, nasal, oral or nasolacrimal delivery. This        document describes that the presence of alkyl glycosides leads        to an improvement of the absorption of human glucagon at the        ocular site,    -   the application WO2012059764 (ARECOR) describes cationic        surfactants and, more precisely, aromatic ammonium chlorides.

The surfactants indicated in the above documents can be too toxic orirritating for chronic use by the subcutaneous route. For example, thelysophospholipids (or lysolecithins) are known to lyse the red bloodcells due to their hemolytic properties. In a subcutaneous injection,this can cause local damage to the tissues and pains at the injectionsite. In the case of continuous injection by means of a pump, this canlead to pains and/or irritation at the site of insertion of the needle.The international application WO2011138802 (Sun Pharma) describes aready-to-use solution of human glucagon in a micellar aqueous solutionat a pH from 5 to 7.5 in the presence of a pegylated lipid (pegylateddistearoylphosphatidylethanolamine). However, Garay et al. (Expert OpinDrug Deliv (2012) 9, 1319-1323) teach that polyethylene glycol is bothimmunogenic and antigenic. This may be prejudicial to patients withanti-PEG antibodies. Moreover, Ganson et al. (J. Allergy Clin. Immunol.(2015) doi: 10.1016/j.jaci.2015.10.034) describe that a clinical studypertaining to pegnivacogin coupled to 40 kDa methoxypolyethylene glycol(mPEG) led to inflammatory responses starting with the first dose ofpegnivacogin in 3 of the 640 patients. Among these three patients, twomet the anaphylaxis criteria, and one had an isolated dermal reaction;each event was considered serious, and one was even considered lifethreatening to the patient. These adverse events resulted in thestopping of the clinical trial and raise the problem of the adverseeffects of pegylated compounds.

The document WO2013101749 (LATITUDE) describes nanoemulsions of humanglucagon. However, relatively moderate performances are claimed in termsof chemical stability, that is to say that the composition comprises atleast 75% of the initial concentration after 3-7 days at 37° C.

In addition, it should be noted that, to this date, to the knowledge ofthe applicant, no pharmaceutical formulation comprising human glucagonin the form of an aqueous solution is being tested in a clinical study.

Thus, a need remains for a liquid aqueous formulation at a pH close tophysiological pH, from 6.0 to 8.0, which enables solubilization and theobtention of a satisfactory stability of human glucagon, both in termsof physical stability and chemical stability. More particularly, thereis need for such a formulation that can be used in a bi-hormonal pump(insulin/human glucagon).

This need is particularly clear in view of the fact that Tan et al.(Diabetes, 2013, 62, 1131-138) shows that combining human glucagon witha GLP-1 RA is an attractive proposition for treating obesity anddiabetes. Now, being able to formulate human glucagon in a stable mannerin an aqueous solution at a pH close to physiological pH from 6.0 to 8.0makes it possible to be under conditions more favorable for being ableto improve the stability of the GLP-1 RA which are sensitive to acidicor alkaline conditions.

The co-polyamino acids bearing carboxylate charges and hydrophobicradicals Hy according to the invention present an excellent resistanceto hydrolysis. This can be shown particularly under acceleratedconditions, for example, in hydrolysis tests at alkaline pH (pH 12).

In addition, forced oxidation tests, for example, of the Fentonoxidation type, show that the co-polyamino acids bearing carboxylatecharges and hydrophobic radicals Hy present a good resistance tooxidation.

The invention thus relates to physically stable compositions in the formof an injectable aqueous solution, the pH of which is from 6.0 to 8.0,comprising at least:

a) human glucagon, and

b) a co-polyamino acid bearing carboxylate charges and hydrophobicradicals Hy, said co-polyamino acid consisting of glutamic or asparticunits, and said hydrophobic radicals Hy being radicals of the followingformula I:

in which

-   -   GpR is a radical of formula II:

-   -   GpA is a radical of formula III or III′:

-   -   GpC is a radical of formula IV:

-   -   the * indicate the sites of attachment of the different groups;    -   a is a whole number equal to 0 or 1;    -   b is a whole number equal to 0 or 1;    -   p is a whole number equal to 1 or 2, and        -   if p is equal to 1, then a is equal to 0 or 1 and GpA is a            radical of formula III′, and        -   if p is equal to 2, then a is equal to 1 and GpA is a            radical of formula III;    -   c is a whole number equal to 0 or 1, and, if c is equal to 0,        then d is equal to 1 or 2;    -   d is a whole number equal to 0, to 1 or 2;    -   r is a whole number equal to 0 or 1, and        -   if r is equal to 0, then the hydrophobic radical of formula            I is bound to the co-polyamino acid via a covalent bond            between a carbonyl of the hydrophobic radical and a nitrogen            atom in N-terminal position of the co-polyamino acid            therefore forming an amide function originating from the            reaction of an amine function in N-terminal position of the            precursor of the co-polyamino acid and an acid function            borne by the precursor of the hydrophobic radical, and        -   if r is equal to 1, then the hydrophobic radical of formula            I is bound to the co-polyamino acid:            -   via a covalent bond between a nitrogen atom of the                hydrophobic radical and a carbonyl of the co-polyamino                acid therefore forming an amide function originating                from the reaction between an amine function of the                precursor of the hydrophobic radical and an acid                function borne by the precursor of the co-polyamino                acid, or            -   via a covalent bond between a carbonyl of the                hydrophobic radical and a nitrogen atom in N-terminal                position of the co-polyamino acid therefore forming an                amide function originating from the reaction of an acid                function of the precursor of the hydrophobic radical and                an amine function in N terminal position borne by the                precursor of the co-polyamino acid;    -   R is a radical selected from the group consisting of:        -   a linear or branched divalent alkyl radical comprising, if            GpR is a radical of formula II, from 2 to 12 carbon atoms;        -   a linear or branched divalent alkyl radical comprising, if            GpR is a radical of formula II, from 2 to 11 carbon atoms,            said alkyl radical bearing one or more —CONH2 functions, and        -   an unsubstituted ether or polyether radical comprising from            4 to 14 carbon atoms and from 1 to 5 oxygen atoms;    -   A is a linear or branched alkyl radical comprising from 1 to 6        carbon atoms;    -   B is a linear or branched alkyl radical, optionally comprising        an aromatic ring, comprising from 1 to 9 carbon atoms;    -   C_(x) is a linear or branched monovalent alkyl radical, in which        x indicates the number of carbon atoms, and:        -   if p is equal to 1, x is from 11 to 25 (11≦x≦25);        -   if p is equal to 2, x is from 9 to 15 (9≦x≦15),    -   the ratio i between the number of hydrophobic radicals and the        number of glutamic or aspartic units being between 0<i≦0.5;    -   when several hydrophobic radicals are borne by a co-polyamino        acid, then they are identical or different,    -   the degree of polymerization DP in glutamic or aspartic units is        from 5 to 250;    -   the free acid functions being in the form of a salt of an        alkaline cation selected from the group consisting of Na⁺ and        K⁺.

In an embodiment, the composition is characterized in that the pH isfrom 6.6 to 7.8.

In an embodiment, the composition is characterized in that the pH isfrom 7.0 to 7.8.

In an embodiment, the composition is characterized in that the pH isfrom 6.8 to 7.4.

In an embodiment, the composition is characterized in that saidhydrophobic radicals are selected from the hydrophobic radicals offormula I in which p is equal to 1, and, if x is less than or equal to14 (x≦14), then r=0 or r=1.

In an embodiment, the composition is characterized in that saidhydrophobic radicals are selected from the hydrophobic radicals offormula I in which p is equal to 1, and, if x is from 15 to 16(15≦x≦16), then r=1.

In an embodiment, the composition is characterized in that saidhydrophobic radicals are selected from the hydrophobic radicals offormula I in which p is equal to 1, and, if x is greater than 17 (17≦x),then r=1 and R is an ether or polyether radical.

In an embodiment, the composition is characterized in that saidhydrophobic radicals are selected from the hydrophobic-radicals offormula I in which, if p is equal to 1, then x is from 17 to 25(17≦x≦25).

In an embodiment, the composition is characterized in that saidhydrophobic radicals are selected from the hydrophobic radicals offormula I in which p=1, represented by the following formula V:

GpR, GpA, GpC, r and a have the definitions given above.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V, in which r is equal to 1(r=1), and a is equal to 0 (a=0).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which r is equal to 1(r=1) and a is equal to 1 (a=1).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II, in which R is a divalent linear alkyl radical comprisingfrom 2 to 12 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula Vin which GpR is a radicalof formula II in which R is a divalent alkyl radical comprising from 2to 6 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II in which R is a divalent linear alkyl radical comprisingfrom 2 to 6 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II in which R is a divalent alkyl radical comprising from 2to 4 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II in which R is a divalent linear alkyl radical comprisingfrom 2 to 4 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II in which R is a divalent alkyl radical comprising 2 carbonatoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II′.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II in which R is a divalent alkyl radical comprising from 2to 5 carbon atoms and bearing one or more amide functions (—CONH₂).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II, in which R is a divalent linear alkyl radical comprisingfrom 2 to 5 carbon atoms and bearing one or more amide functions(—CONH₂).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II in which R is a radical selected from the group consistingof the radicals represented by the formulas below:

In an embodiment, the composition is characterized in that the radical Ris bound to the co-polyamino acid via an amide function borne by thecarbon in delta or epsilon position (or in position 4 or 5) with respectto the amide function (—CONH₂).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II or II′, in which R is an unsubstituted linear ether orpolyether radical comprising from 4 to 14 carbon atoms and from 1 to 5oxygen atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II or II′, in which R is an ether radical.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II or II′, in which R is an ether radical comprising from 4to 6 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II, in which R is an ether radical represented by the formula

of formula X7.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II or II′, in which R is a polyether radical.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which GpR is a radicalof formula II or II′, in which R is a linear polyether radicalcomprising from 6 to 10 carbon atoms and from 2 to 3 oxygen atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIor II′, in which R is a polyether radical selected from the groupconsisting of the radicals represented by the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIor IP, in which R is a radical of formula X3.

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIor II′, in which R is a radical of formula X4.

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIor II′, in which R is a radical of formula X5.

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIor II′, in which R is a radical of formula X6.

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIin which R is a polyether radical selected from the group consisting ofthe radicals represented by the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIin which R is a polyether radical of formula X5.

In an embodiment, the composition is characterized in that thehydrophobic radical of formula V in which GpR is a radical of formula IIin which R is a polyether radical of formula X6.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 0(a=0) and r is equal to 0 (r=0).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1), and the radical GpA of formula III′ is selected from the groupconsisting of the radicals represented by the formulas below;

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1), and the radical GpA of formula III′ is a radical of formula Y1.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1), and the radical GpA of formula III′ is a radical of formula Y2.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1) and the radical GpA of formula III′ is a radical of formula Y3.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1) and the radical GpA of formula III′ is a radical of formula Y4.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1) and the radical GpA of formula III′ is a radical of formula Y5.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1) and the radical GpA of formula III′ is a radical of formula Y6.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1) and the radical GpA of formula III′ is a radical of formula Y7.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which a is equal to 1(a=1) and the radical GpA of formula III′ is a radical of formula Y8.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals offormula IVa, IVb or IVc represented hereafter:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCis of formula IVa.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals offormula IVa, IVb or IVc in which b is equal to 0, respectivelycorresponding to formulas IVd, IVe and IVf, represented hereafter:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCcorresponds to formula IV or IVa in which b=0, and corresponds toformula IVd.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV in which b=1 is selected from the group consisting of theradicals in which B is an amino acid residue selected from the groupconsisting of the radicals represented by the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula corresponds to formula IV or IVa in which b=1, is selectedfrom the group consisting of the radicals in which B is an amino acidresidue selected from the group consisting of the radicals representedby the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the linear alkylradicals.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of radicals in whichCx is selected from the group consisting of the branched alkyl radicals.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of alkyl radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising from 11 to 14 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of radicals in whichCx is selected from the group consisting of the radicals represented bythe formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising from 15 to 16 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the radicalsrepresented by the formulas below;

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the radicalsrepresented by the formulas below:

In an embodiment, the composition is characterized that the hydrophobicradical is a radical of formula V in which the radical GpC of formula IVis selected from the group consisting of the radicals in which Cx isselected from the group consisting of the alkyl radicals comprising from17 to 25 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising from 17 to 18 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalsrepresented by the formulas below:

In an embodiment, the composition characterized in that the hydrophobicradical is a radical of formula V in which the radical GpC of formula IVis selected from the group consisting of the radicals in which Cx isselected from the group consisting of the alkyl radicals comprising from18 to 25 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula V in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalsrepresented by the formulas below:

In an embodiment, the composition is characterized in that saidhydrophobic radicals of formula I are selected from the hydrophobicradicals of formula I in which a=1 and p=2, represented by the followingformula VI:

in which

GpR, GpA, GpC, r and a have the definitions given above.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which r=1.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a divalent linear alkyl radical comprisingfrom 2 to 12 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a divalent alkyl radical comprising from 2to 6 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula U in which R is a divalent linear alkyl radical comprisingfrom 2 to 6 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is an alkyl radical comprising from 2 to 4carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a divalent linear alkyl radical comprisingfrom 2 to 4 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a divalent linear alkyl radical comprising 2carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula IF.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II, in which R is a divalent alkyl radical comprising from 2to 5 carbon atoms and bearing one or more amide functions (—CONH2).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II, in which R is a divalent linear alkyl radical comprisingfrom 2 to 5 carbon atoms and bearing one or more amide functions(—CONH2).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II, in which R is a radical selected from the groupconsisting of the radicals represented by the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the aminefunction of the GpR radical involved in the formation of the amidefunction which binds said GpR radical to the co-polyamino acid is borneby a carbon in delta or epsilon position (or in position 4 or 5) withrespect to the amide function (—CONH2).

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II, in which R is an unsubstituted linear ether or polyetherradical comprising from 4 to 14 carbon atoms and from 1 to 5 oxygenatoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is an ether radical.

In an embodiment, the composition is characterized in that the etherradical R is a radical comprising from 4 to 6 carbon atoms.

In an embodiment, the composition is characterized in that the etherradical is

of formula X7.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II, in which R is a polyether radical.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II, in which R is a linear polyether radical comprising from6 to 10 carbon atoms and from 2 to 3 oxygen atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a linear polyether radical selected from thegroup consisting of the radicals represented by the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a linear polyether radical of formula X4.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a linear polyether radical of formula X5.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which GpR is a radicalof formula II in which R is a linear polyether radical of formula X6.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpAof formula III is selected from the group consisting of the radicals offormulas IIIa, IIIb and IIIc represented hereafter:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpAof formula II is a radical of formula IIIb represented hereafter:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpAof formula III is a radical of formula IIIc.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals offormulas IVa, IVb and IVc represented hereafter:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCis of formula IVa.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals offormula IVa, IVb or IVc in which b is equal to 0, respectivelycorresponding to the formulas IVd, IVe and IVf, represented hereafter:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCcorresponds to formula IV or IVa in which b=0, and it corresponds toformula IVd.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the linear alkylradicals comprising from 9 to 15 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the branched alkylradicals comprising from 9 to 15 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising 9 or 10 carbon atoms.

in an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising from 11 to 15 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising from 11 to 13 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the radicalsrepresented by the formulas below:

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the alkyl radicalscomprising 14 or 15 carbon atoms.

In an embodiment, the composition is characterized in that thehydrophobic radical is a radical of formula VI in which the radical GpCof formula IV is selected from the group consisting of the radicals inwhich Cx is selected from the group consisting of the radicalsrepresented by the formulas below:

The co-polyamino acid bearing carboxylate charges and at least onehydrophobic radical of formula I can also be referred to as“co-polyamino acid” in the present description.

In an embodiment, the composition is characterized in that theco-polyamino acid bearing carboxylate charges and hydrophobic radicalsis selected from the co-polyamino acids of the following formula VIIa:

in which,

-   -   D represents, independently, either a —CH₂— group (aspartic        unit) or a —CH₂—CH₂— group (glutamic unit),    -   Hy is a hydrophobic radical selected from the hydrophobic        radicals of formula I, V or VI, in which r=1 and GpR is a        radical of Formula II,    -   X represents H or a cationic entity selected from the group        comprising the metal cations;    -   n+m represents the degree of polymerization DP of the        co-polyamino acid, that is to say the average number of monomer        units per co-polyamino acid chain and 5≦n+m≦250;        R′1 is a radical selected from the group consisting of H, a C2        to C10 linear acyl group, a C4 to C10 branched acyl group,        benzyl, a terminal “amino acid” unit and a pyroglutamate,    -   R′2 is a —NR′R″ radical, R′ and R″, which are identical or        different, being selected from the group consisting of H, the C2        to C10 linear or branched or cyclic alkyls, benzyl, and said        alkyl R′ and R″ together optionally forming one or more        saturated, unsaturated and/or aromatic carbon rings and/or        optionally comprising heteroatoms selected from the group        consisting of O, N and S.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is a radical of formula V or of formula VI,with r=1.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is a radical of formula V or of formula VI,with GpR of formula II.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is a radical of formula V.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula V and GpC is a radical of formulaIVd.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula V and GpC is a radical of formulaIVd in which x=13.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula V and GpC is a radical of formulaIVd in which x=15.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula V and GpC is a radical of formulaIVd in which x=17.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula V and GpC is a radical of formulaIVd in which x=19.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is a radical of formula VI.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is a radical of formula VI in which r=1 and GpRis of formula I.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is a radical of formula VI in which r=1 and GpRis of formula II and for GpC, b=0.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula VI, GpR of formula II and GpC isa radical of formula IVd.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula VI, GpR of formula II and GpC isa radical of formula IVd and r=1.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid bearing carboxylate chargesand hydrophobic radicals is selected from the co-polyamino acids offormula VIIa in which Hy is of formula VI, GpR of formula II and GpC isa radical of formula IVd in which x is from 11 to 15.

In an embodiment, the composition according to the invention ischaracterized in that, when the co-polyamino acid comprises aspartateunits, then the co-polyamino acid can, in addition, comprise monomerunits of formula VIII and/or VIII′:

In an embodiment, the composition is characterized in that R₁ is aradical selected from the group consisting of a C₂ to C₁₀ linear acylgroup, a C₄ to C₁₀ branched acyl group, benzyl, a terminal “amino acid”unit and a pyroglutamate.

In an embodiment, the composition is characterized in that R₁ is aradical selected from the group consisting of a C₂ to C₁₀ linear acylgroup or a C₄ to C₁₀ branched acyl group.

In an embodiment, the composition is characterized in that theco-polyamino acid bearing carboxylate charges and hydrophobic radicalsis selected from the co-polyamino acids of formula VIIa in which thegroup D is a group —CH₂— (aspartic unit).

In an embodiment, the composition is characterized in that theco-polyamino acid bearing carboxylate charges and hydrophobic radicalsis selected from the co-polyamino acids of formula VIIa in which thegroup D is a —CH₂—CH₂— group (glutamic unit).

In an embodiment, the composition is characterized in that the ratio ibetween the number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.007 to 0.3.

In an embodiment, the composition is characterized in that the ratio ibetween the number of hydrophobic radicals and the number of glutamic ofglutamic or aspartic units is from 0.01 to 0.3.

In an embodiment, the composition is characterized in that the ratio ibetween the number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.02 to 0.2.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI and the ratio i betweenthe number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.007 to 0.15.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI and the ratio i betweenthe number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.01 to 0.1.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI and the ratio i betweenthe number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.02 to 0.08.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI in which the radical Cxcomprises from 9 to 10 carbon atoms and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.03 to 0.15.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI in which the radical Cxcomprises from 11 to 12 carbon atoms and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.015 to 0.1.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI in which the radical Cxcomprises from 11 to 12 carbon atoms and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.02 to 0.08.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI in which the radical Cxcomprises from 13 to 15 carbon atoms and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.01 to 0.1.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula VI in which the radical Cxcomprises from 13 to 15 carbon atoms, and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.01 to 0.06.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V, and the ratio i betweenthe number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.007 to 0.3.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V, and the ratio i betweenthe number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.01 to 0.3.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V, and the ratio i betweenthe number of hydrophobic radicals and the number of glutamic oraspartic units is from 0.015 to 0.2.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V in which the radical Cxcomprises from 11 to 14 carbon atoms, and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.1 to 0.2.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V in which the radical Cxcomprises from 15 to 16 carbon atoms, and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.04 to 0.15.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V in which the radical Cxcomprises from 17 to 18 carbon atoms, and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.02 to 0.06.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V in which the radical Cxcomprises from 19 to 25 carbon atoms, and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.01 to 0.06.

In an embodiment, the composition is characterized in that thehydrophobic radical corresponds to formula V in which the radical Cxcomprises from 19 to 25 carbon atoms, and the ratio i between the numberof hydrophobic radicals and the number of glutamic or aspartic units isfrom 0.01 to 0.05.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 10 to 250.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 10 to 200.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 15 to 150.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 15 to 100.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 15 to 80.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 15 to 65.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 20 to 60.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 20 to 50.

In an embodiment, the composition according to the invention ischaracterized in that n+m is from 20 to 40.

The invention also relates to said co-polyamino acid bearing carboxylatecharges and hydrophobic radicals of formula I and the precursors of saidhydrophobic radicals.

The co-polyamino acid bearing carboxylate charges and hydrophobicradicals of formula I are soluble in distilled water at a pH from 6 to8, at a temperature of 25° C., and at a concentration of less than 100mg/mL.

The invention moreover relates to a method for preparing stableinjectable compositions.

“Soluble” is understood to mean capable of enabling the preparation of aclear, particle-free solution at a concentration of less than 100 mg/mLin distilled water at 25° C.

“Solution” is understood to mean a liquid composition free of visibleparticles, using the procedure according to the pharmacopoeias EP 8.0,under point 2.9.20, and US<790>.

“Physically stable composition” is understood to mean compositionswhich, after a certain storage time at a certain temperature meet thecriteria of visual inspection described in the European, American andinternational pharmacopoeias, that is to say compositions which areclear and contain no visible particles, and are also colorless.

“Chemically stable composition” is understood to mean compositions whichafter a certain storage time at a certain temperature, present a minimumrecovery of the active ingredients and are in compliance with thespecifications applicable to the pharmaceutical products.

A conventional method for measuring the stabilities of the proteins orpeptides consists in measuring the formation of fibrils with the aid ofThioflavin T, also referred to as ThT. This method makes it possible,under temperature and stirring conditions that enable an acceleration ofthe phenomenon, to measure the lag time before the formation of fibrilsby measuring the increase in fluorescence. The compositions according tothe invention have a lag time before the formation of fibrils which isclearly greater than the lag time of the glucagon at the pH of interest.

“Injectable aqueous solution” is understood to mean water-basedsolutions which meet the conditions of the EP and US pharmacopoeias, andwhich are sufficiently liquid to be injected.

“Co-polyamino acid consisting of glutamic or aspartic units” isunderstood to mean noncyclic linear chains of glutamic acid or asparticacid units bound together by peptide bonds, said chains having aC-terminal part corresponding to the carboxylic acid of one end and anN-terminal part corresponding to the amine of the other end of thechain.

“Alkyl radical” is understood to mean a linear or branched carbon chainwhich comprises no heteroatom.

The co-polyamino acid is a statistical or block co-polyamino acid.

The co-polyamino acid is a statistical co-polyamino acid in the chain ofthe glutamic and/or aspartic units.

In the formulas, the * indicate the sites of attachment of the differentelements represented.

In formulas I, V and VI, the * indicate the sites of attachment of thehydrophobic radicals to the co-polyamino acid. The radicals Hy areattached to the polyamino acid via amide functions.

In formulas II and II′, the * indicate, from left to right,respectively, the sites of attachment of GpR:

-   -   to the co-polyamino acid and    -   to the GpA if a=1, or to GPC if a=0.

In formulas III and III′, the * indicate, from left to right,respectively, the sites of attachment of GpA:

-   -   to GpR if r=1, or to the co-polyamino acid if r=0, and    -   to GpC.

In formula IV, the * indicates the site of attachment of GpC:

-   -   to GpA if a=1, GpR if r=1 and a=0, or to the co-polyamino acid        if r=0 and a=0.

All the attachments between the different groups GpR, GpA and GpC areamide functions.

The radicals Hy, GpR, GpA, GpC, and D are each independently identicalor different from one monomer unit to the next.

When the co-polyamino acid comprises one or more aspartic unit(s), it(they) can undergo structural rearrangements.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acids obtained moreover comprisemonomer units of formula VIII and/or VIII′:

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which, r=1, a=0, p=1, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpC corresponds toformula IVd in which x=15 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which, r=1, a=0, p=1, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpC corresponds toformula IVd in which x=16 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is

GpC corresponds to formula IVd in which x=15 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is

GpC corresponds to formula IVd in which x=15 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula 1 in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is

GpC corresponds to formula IVd in which x=15 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is

GpC corresponds to formula IVd in which x=17 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is

GpC corresponds to formula IVd in which x=19 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpC corresponds toformula IVa in which b=1, B is

x=15 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpC corresponds toformula IVa in which b=1, B is

x=11 and Cx

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula 1 in which r=1, a=1, p=2, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpA corresponds toformula IIIb, GpC corresponds to formula IVd in which x=9 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=1, p=2, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpA corresponds toformula IIIb, GpC corresponds to formula IVd in which x=11 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=1, p=2, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpA corresponds toformula IIIb, GpC corresponds to formula IVd in which x=13 and Cx is

In an embodiment, the at least one hydrophobic radical of formula I isselected from the radicals of formula I in which r=1, a=1, p=2, GpRcorresponds to formula II in which R is

GpA corresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=23+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpCcorresponds to formula IVd in which x=15 and Cx is

The values of the degree of polymerization DP and of ratio i areestimated by ¹H NMR in D₂O by comparing the integration of the signalsoriginating from the hydrophobic groups with the integration of thesignals originating from the main chain of the co-polyamino acid.

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=35+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula is selected from the radicals of formula I in which r=1, a=0,p=1, GpR corresponds to formula II in which R equals —CH₂—CH₂—, GpCcorresponds to formula IVd in which x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=35+/−5, i=0.10+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R equals —CH₂—CH₂—, GpCcorresponds to formula IVd in which x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=35+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R equals —CH₂—CH₂—, GpCcorresponds to formula IVd in which x=16 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=23+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=17 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VU or VIIa inwhich DP=22+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=19 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=30+/−5, i=0.10+/−0.03 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=23+/−5, i=0.07+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=23+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=26+/−5, i=0.04+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpCcorresponds to formula IVa in which b=1, B is

x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=35+/−5, i=0.13+/−0.04 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpCcorresponds to formula IVa in which b=1, B is

x=11 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa, inwhich DP=23+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=19 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=25+/−5, 0.033≦i≦0.05, and the hydrophobic radical of formula Iis selected from the radicals of formula I in which r=1, a=0, p=1, GpRcorresponds to formula II in which R is —CH₂—CH₂—, GpC corresponds toformula IVd in which x=15 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=30+/−5, 0.028≦i≦0.04, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=17 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=45+/−10, 0.018≦i≦0.028, and the at least one hydrophobicradical of formula I is selected from the radicals of formula f in whichr=1, a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=17 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=60+/−10, 0.014≦i≦0.02, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=0, p=1, GpR corresponds to formula II in which R is

GpC corresponds to formula IVd in which x=17 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=25+/−5, 0.033≦i≦0.05, and the hydrophobic radical of formula Iis selected from the radicals of formula I in which r=1, a=0, p 1=, GpRcorresponds to formula II in which R is

GpC corresponds to formula IVd in which x=19 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=22+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula fl in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=11 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=35+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=11 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=65+/−5, i=0.05+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=11 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=22+/−5, i=0.04+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=22+/−5, 0.03+/−0.01 and the at least one hydrophobic radical offormula I is selected from the radicals of formula I in which r=1, a=1,p=2, GpR corresponds to formula II in which R is

GpA corresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIa inwhich DP=22+/−5, i=0.07+/−0.02 and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is

GpA corresponds to formula IIIb, GpC corresponds to formula IVd in whichx=9 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=27+/−5, 0.031≦i≦0.045, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=11 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=22+/−5, 0.037≦i≦0.055, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=22+/−5, 0.037≦i≦0.055, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is

GpA corresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=60+/−10, 0.014≦i≦0.02, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, GpAcorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is a co-polyamino acid of formula VII or VIIb, inwhich DP=40+/−5, 0.022≦i≦0.029, and the at least one hydrophobic radicalof formula I is selected from the radicals of formula I in which r=1,a=1, p=2, GpR corresponds to formula II in which R is —CH₂—CH₂—, Acorresponds to formula IIIb, GpC corresponds to formula IVd in whichx=13 and Cx is

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by ring-opening polymerization of a glutamic acidN-carboxyanhydride derivative or an aspartic acid N-carboxyanhydridederivative.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization of a glutamic acid N-carboxyanhydridederivative or of an aspartic acid N-carboxyanhydride derivativedescribed in the review article Adv. Polym. Sci. 2006, 202, 1-18(Deming, T. J.).

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization of a glutamic acid N-carboxyanhydridederivative.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization of a glutamic acid N-carboxyanhydridederivative selected from the group consisting of methyl glutamateN-carboxyanhydride (GluOMe-NCA), benzyl glutamate N-carboxyanhydride(GluOBzl-NCA), and t-butyl glutamate N-carboxyanhydride (GluOtBu-NCA).

In an embodiment, the glutamic acid N-carboxyanhydride derivative ismethyl L-glutamate N-carboxyanhydride (L-GluOMc-NCA).

In an embodiment, the glutamic acid N-carboxyanhydride derivative isbenzyl L-glutamate N-carboxyanhydride (L-GluOBzl-NCA).

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization of a glutamic acid N-carboxyanhydridederivative or of an aspartic acid N-carboxyanhydride derivative using,using as initiator, an organometallic complex of a transition metal, asdescribed in the publication Nature 1997, 390, 386-389 (Deming, TJ.).

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization of a glutamic acid N-carboxyanhydridederivative or of an aspartic acid N-carboxyanhydride derivative, using,as initiator, ammonia or a primary amine as described in the patent FR2,801,226 (Touraud, F. et al.) and the references cited in this patent.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by polymerization of a glutamic acid N-carboxyanhydridederivative or of an aspartic acid N-carboxyanhydride derivative, usingas initiator hexamethyldisilazane, as described in the publication J.Am. Chem. Soc. 2007, 129, 14114-14115 (Lu H. et al.) or a silylatedamine as described in the publication J. Am. Chem. Soc. 2008, 130,12562-12563 (Lu H. et al.).

In an embodiment, the composition according to the invention ischaracterized in that the method for synthetizing the polyamino acidobtained by polymerization of a glutamic acid N-carboxyanhydridederivative or an aspartic acid N-carboxyanhydride derivative from whichthe co-polyamino acid originates comprises a step of hydrolysis of esterfunctions.

in an embodiment, this step of hydrolysis of ester functions can consistof a hydrolysis in an acidic medium or a hydrolysis in an alkalinemedium or it can be carried out by hydrogenation.

In an embodiment, this step of hydrolysis of ester groups is ahydrolysis in an acidic medium.

In an embodiment, this step of hydrolysis of ester groups is carried outby hydrogenation.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by depolymerization of a polyamino acid of highermolecular weight.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by enzymatic depolymerization of a polyamino acid ofhigher molecular weight.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by chemical depolymerization of a polyamino acid of highermolecular weight.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by enzymatic and chemical depolymerization of a polyaminoacid of higher molecular weight.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by depolymerization of a polyamino acid of highermolecular weight selected from the group consisting of sodiumpolyglutamate and sodium polyaspartate.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by depolymerization of a sodium polyglutamate of highermolecular weight.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid originates from a polyaminoacid obtained by depolymerization of a sodium polyaspartate of highermolecular weight.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid is obtained by grafting ahydrophobic group onto a poly-L-glutamic acid or poly-L-aspartic acidusing the methods for forming amide bonds, which are well known to theperson skilled in the art.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid is obtained by grafting of ahydrophobic group onto a poly-L-glutamic acid or poly-L-aspartic acidusing the methods for forming amide bonds, used for peptide synthesis.

In an embodiment, the composition according to the invention ischaracterized in that the co-polyamino acid is obtained by grafting of ahydrophobic group onto a poly-L-glutamic acid or poly-L-aspartic acid asdescribed in the patent FR 2,840,614 (Chan, Y. P.; et al.).

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 40 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 30 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 20 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 10 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 5 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 2.5 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 1 mg/mL.

In an embodiment, the concentration of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is at most 0.5 mg/mL.

In an embodiment, the weight ratio of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals over glucagon is from 1.5to 25.

In an embodiment, the weight ratio of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals over glucagon is from 2 to20.

In an embodiment, the weight ratio of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals over glucagon is from 2.5to 15.

In an embodiment, the weight ratio of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals over glucagon is from 2 to10.

In an embodiment, the weight ratio of co-polyamino acid bearingcarboxylate charges and hydrophobic radicals over glucagon is from 2 to7.

Human glucagon is used at doses that vary depending on the applications.

In the emergency treatment of hypoglycemia, the recommended dosage is 1mg by the intramuscular or intravenous route (0.5 mg if the body weightis less than 25 kg). This administration is carried out with a solutionof human glucagon at the concentration of 1 mg/mL.

In pumps, the daily dose considered is approximately 0.5 mg; thus thesolutions can comprise from 0.25 mg/mL to 5 mg/mL of human glucagon.

In an embodiment, the solutions can comprise from 0.5 mg/mL to 3 mg/mLof human glucagon.

In the treatment of obesity, the daily dose considered is approximately0.5 mg, the solutions thus can comprise from 0.25 mg/mL to 5 mg/mL ofhuman glucagon.

In an embodiment, the concentration of human glucagon is from 0.25 to 5mg/mL.

In an embodiment, the concentration of human glucagon is from 0.5 to 4mg/mL.

In an embodiment, the concentration of human glucagon is from 0.75 to 3mg/mL.

In an embodiment, the concentration of human glucagon is from 0.75 to2.5 mg/mL.

In an embodiment, the concentration of human glucagon is from 0.75 to 2mg/mL.

In an embodiment, the concentration of human glucagon is from 1 to 2mg/mL.

In an embodiment, the molar ratio [hydrophobic radical]/[human glucagon]is less than 20.

In an embodiment, the molar ratio [hydrophobic radical]/[human glucagon]is less than 15.

In an embodiment, the molar ratio [hydrophobic radical]/[human glucagon]is less than 10.

In an embodiment, the molar ratio [hydrophobic radical]/[human glucagon]is less than 5.

In an embodiment, the molar ratio [hydrophobic radical]/[human glucagon]is less than 2.5.

In an embodiment, the molar ratio [hydrophobic radical]/[human glucagon]is less than 1.5.

Human glucagon is a highly preserved polypeptide comprising a simplechain of 29 amino acid residues having the following sequenceH-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gn-Asp-Phe-Val-Gn-Trp-Leu-Met-Asn-Thr-OH.

It can be obtained in different manners, by recombinant peptidesynthesis.

Human glucagon is available from numerous sources. For example, thehuman glucagon produced by Bachem via peptide synthesis is available, inparticular under reference 407473.

In an embodiment, the composition comprises, in addition, a nicotiniccompound or one of the derivatives thereof.

In an embodiment, the composition comprises nicotinamide.

In an embodiment, the concentration of nicotinamide ranges from 10 to160 mM.

In an embodiment, the concentration of nicotinamide ranges from 20 to150 mM.

In an embodiment, the concentration of nicotinamide ranges from 40 to120 mM.

In an embodiment, the concentration of nicotinamide ranges from 60 to100 mM.

In an embodiment, the composition comprises, in addition, a polyanioniccompound

In an embodiment, the polyanionic compound is selected from the groupconsisting of the carboxylic polyacids and the Na⁺, K⁺, Ca²⁺ or Mg²⁺salts thereof.

In an embodiment, the carboxylic acid is selected from the groupconsisting of citric acid, tartaric acid and the Na⁺, K⁺, Ca²⁺ or Mg²⁺salts thereof.

In an embodiment, the polyanionic compound is selected from the groupconsisting of the phosphoric polyacids and the Na⁺, K⁺, Ca²⁺ or Mg²⁺salts thereof.

In an embodiment, the phosphoric polyacid is triphosphate and the Na⁺,K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In an embodiment, the polyanionic compound is citric acid and the Na⁺,K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In an embodiment, the polyanionic compound is tartaric acid and the Na⁺,K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In an embodiment, the polyanionic compound is triphosphoric acid and theNa⁺, K⁺, Ca²⁺ or Mg²⁺ salts thereof.

In an embodiment, the concentration of polyanionic compound is from 1 to20 mM.

In an embodiment, the concentration of polyanionic compound is from 2 to15 mM.

In an embodiment, the concentration of polyanionic compound is from 3 to12 mM.

In an embodiment, the concentration of polyanionic compound is 10 mM.

In an embodiment, the concentration of polyanionic compound is 5 mM.

In an embodiment, the concentration of polyanionic compound is 10 mM forconcentrations of glucagon from 0.5 mg/mL to 3 mg/mL.

In an embodiment, the concentration of polyanionic compound is 10 mM forconcentrations of glucagon from 0.5 mg/mL to 2 mg/mL.

In an embodiment, the concentration of polyanionic compound is 10 mM forconcentrations of glucagon from 1 mg/mL to 2 mg/mL.

In an embodiment, the concentration of polyanionic compound is 5 mM forconcentrations of glucagon from 0.5 mg/mL to 3 mg/mL.

In an embodiment, the concentration of polyanionic compound is 5 mM forconcentrations of glucagon from 0.5 mg/mL to 2 mg/mL.

In an embodiment, the concentration of polyanionic compound is 5 mM forconcentrations of glucagon from 1 mg/mL to 2 mg/mL.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is from 1 to 20 mM.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is from 2 to 15 mM.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is from 3 to 12 mM.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 10 mM.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 5 mM.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 10 mM for concentrations of glucagon from 0.5mg/mL to 3 mg/mL.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 10 mM for concentrations of glucagon from 0.5mg/mL to 2 mg/mL.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 10 mM for concentrations of glucagon from 1mg/mL to 2 mg/mL.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 5 mM for concentrations of glucagon from 0.5mg/mL to 3 mg/mL.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 5 mM for concentrations of glucagon from 0.5mg/mL to 2 mg/mL.

In an embodiment, the concentration of citric acid and the Na⁺, K⁺, Ca²⁺or Mg²⁺ salts thereof is 5 mM for concentrations of glucagon from 1mg/mL to 2 mg/mL.

In an embodiment, the compositions according to the invention comprise,in addition, a gastrointestinal hormone.

“Gastrointestinal hormones” is understood to mean the hormones selectedfrom the group consisting of the GLP-1 RA for agonists of the humanglucagon-like peptide-1 receptor (glucagon like peptide-1 receptoragonist) and GIP (glucose-dependent insulinotropic peptide),oxyntomodulin (a derivative of human proglucagon), peptide YY, amylin,cholecystokinin, pancreatic polypeptide (PP), ghrelin and enterostatin,the analogs or derivatives thereof and/or the pharmaceuticallyacceptable salts thereof.

In an embodiment, the gastrointestinal hormones are analogs orderivatives of GLP-1 RA (Glucagon like peptide-1 receptor agonist)selected from the group consisting of exenatide or Byetta®(ASTRA-ZENECA), liraglutide or Victoza® (NOVO NORDISK), lixisenatide orLyxumia® (SANOFI), albiglutide or Tanzeum® (GSK) or dulaglutide orTrulicity® (ELI LILLY & CO), the analogs or derivatives thereof and thepharmaceutically acceptable salts thereof.

In an embodiment, the gastrointestinal hormone is pramlintide or Symlin®(ASTRA-ZENECA).

In an embodiment, the gastrointestinal hormone is exenatide or Byetta®,the analogs or derivatives thereof and the pharmaceutically acceptablesalts thereof.

In an embodiment, the gastrointestinal hormone is liraglutide orVictoza®, the analogs or derivatives thereof and the pharmaceuticallyacceptable salts thereof.

In an embodiment, the gastrointestinal hormone is lixisenatide orLyxumia®, the analogs or derivatives thereof and the pharmaceuticallyacceptable salts thereof.

In an embodiment, the gastrointestinal hormone is albiglutide orTanzeum®, the analogs or derivatives thereof and the pharmaceuticallyacceptable salts thereof.

In an embodiment, the gastrointestinal hormone is dulaglutide orTrulicity®, the analogs or derivatives thereof and the pharmaceuticallyacceptable salts thereof.

In an embodiment, the gastrointestinal hormone is pramlintide orSymlin®, the analogs or derivatives thereof and the pharmaceuticallyacceptable salts thereof.

“Analog,” when used in reference to a peptide or a protein, isunderstood to mean a peptide or a protein in which one or moreconstitutive residues of amino acids have been substituted by otherresidues of amino acids and/or in which one or more constitutiveresidues of amino acids have been eliminated and/or in which one or moreconstitutive residues of amino acids have been added. The admissiblepercentage of homology for the present definition of an analog is 50%.

“Derivative,” when used in reference to a peptide or a protein, isunderstood to mean a peptide or a protein or an analog which has beenchemically modified by a substituent which is not present in the peptideor the protein or the analog of reference, that is to say a peptide or aprotein which has been made by creation of covalent bonds, in order tointroduce substituents.

In an embodiment, the substituent is selected from the group consistingof the fatty chains.

In an embodiment, the concentration of gastrointestinal hormone iswithin a range from 0.01 to 10 mg/mL.

In an embodiment, the concentration of exenatide, the analogs orderivatives thereof and the pharmaceutically acceptable salts thereof iswithin a range from 0.04 to 0.5 mg/mL.

In an embodiment, the concentration of liraglutide, the analogs orderivatives thereof and the pharmaceutically acceptable salts thereof iswithin a range from 1 to 10 mg/mL.

In an embodiment, the concentration of lixisenatide, the analogs orderivatives thereof and the pharmaceutically acceptable salts thereof iswithin a range from 0.01 to 1 mg/mL.

In an embodiment, the concentration of pramlintide, the analogs orderivatives thereof and the pharmaceutically acceptable salts thereof iswithin a range from 0.1 to 5 mg/mL.

In an embodiment, the compositions according to the invention areproduced by mixing of human glucagon solutions obtained byreconstitution of lyophilizate and of solutions of GLP-1 RA (Glucagonlike peptide-1 receptor agonist) GLP-1 RA, of analog or of derivative ofGLP-1 RA, said solutions of GLP-1 RA being commercial or reconstitutedfrom lyophilizate.

In an embodiment, the compositions according to the invention comprise,in addition, buffers.

In an embodiment, the compositions according to the invention comprisebuffers at concentrations from 0 to 100 mM.

In an embodiment, the compositions according to the invention comprisebuffers at concentrations from 15 to 50 mM.

In an embodiment, the compositions according to the invention comprise abuffer selected from the group consisting of a phosphate buffer, Tris(trishydroxymethylaminomethane) or sodium citrate.

In an embodiment, the buffer is sodium phosphate.

In an embodiment, the buffer is Tris (trishydroxymethylaminomethane).

In an embodiment, the buffer is sodium citrate.

In an embodiment, the composition comprises, in addition, a zinc salt,in particular zinc chloride.

In an embodiment, the concentration of zinc salt is from 50 to 5000 μM.

In an embodiment, the concentration of zinc salt is from 100 to 2000 μM.

In an embodiment, the concentration of zinc salt is from 200 to 1500 μM.

In an embodiment, the concentration of zinc salt is from 200 to 1000 μM.

In an embodiment, the zinc concentration is such that the molar ratio[zinc]/[glucagon] is from 0.1 to 2.5.

In an embodiment, the zinc concentration is such that the molar ratio[zinc]/[glucagon] is from 0.2 to 2.

In an embodiment, the zinc concentration is such that the molar ratio[zinc]/[glucagon] is from 0.5 to 1.5.

In an embodiment, the zinc concentration is such that the molar ratio[zinc]/[glucagon] is 1.

In an embodiment, the compositions according to the invention comprise,in addition, preservatives.

In an embodiment, the preservatives are selected from the groupconsisting of m-cresol and phenol, alone or in a mixture.

In an embodiment, the compositions according to the invention comprise,in addition, antioxidants.

In an embodiment, the antioxidants are selected from methionine.

In an embodiment, the concentration of preservatives is from 10 to 50mM.

In an embodiment, the concentration of preservatives is from 10 to 40mM.

In an embodiment, the compositions according to the invention comprise,in addition, a surfactant.

In an embodiment, the surfactant is selected from the group consistingof propylene glycol or polysorbate.

The compositions according to the invention can, in addition, compriseadditives such as tonicity agents.

In an embodiment, the tonicity agents are selected from the groupconsisting of sodium chloride, mannitol, saccharose, sorbitol andglycerol.

The compositions according to the invention can comprise, in addition,all the excipients in accordance with the pharmacopoeias and compatiblewith human glucagon and the GLP-1 RA used at the conventionalconcentrations.

The invention also relates to a pharmaceutical composition according tothe invention, characterized in that it is obtained by drying and/orlyophilization.

In the case of local and systemic releases, the modes of administrationconsidered are by intravenous, subcutaneous, intradermal orintramuscular route.

The transdermal, oral, nasal, vaginal, ocular, buccal, pulmonary routesof administration are also considered.

The invention also relates to single-dose formulations at pH from 6.6 to7.8 comprising human glucagon.

The invention also relates to single-dose formulations at a pH from 6.6to 7.8 comprising human glucagon and a gastrointestinal hormone, asdefined above.

In an embodiment, the single-dose formulations comprise, in addition, asubstituted co-polyamino acid as defined above.

In an embodiment, the formulations are in the form of an injectablesolution. In an embodiment, the GLP-1 RA, analog or derivative of GLP-1RA is selected from the group comprising exenatide (Byetta®),liraglutide (Victoza®), lixisenatide (Lyxumia®), albiglutide (Tanzeum®),dulaglutide (Trulicity®) or one of the derivatives thereof.

In an embodiment, the gastrointestinal hormone is exenatide.

In an embodiment, the gastrointestinal hormone is liraglutide.

In an embodiment, the gastrointestinal hormone is lixisenatide.

In an embodiment, the gastrointestinal hormone is albiglutide.

In an embodiment, the gastrointestinal hormone is dulaglutide.

Moreover and just as importantly, the applicant was able to verify thathuman glucagon in the presence of a co-polyamino acid bearingcarboxylate charges and at least one hydrophobic radical according tothe invention preserves its action, whether alone or in combination witha gastrointestinal hormone.

The preparation of a composition according to the invention has theadvantage that it can be carried out by simple mixing of a solution ofhuman glucagon, of a solution of GLP-1 RA, of an analog or a derivativeof GLP-1 RA, and of a co-polyamino acid bearing carboxylate charges andat least one hydrophobic radical according to the invention, in aqueoussolution or in lyophilized form. If necessary, the pH of the preparationis adjusted to pH 7.

In an embodiment, the mixture of human glucagon and of substitutedco-polyamino acid is concentrated by ultrafiltration before the mixingwith GLP-1 RA, an analog or a derivative of GLP-1 RA in aqueous solutionand in lyophilized form.

If necessary, the composition of the mixture is adjusted in terms ofexcipients such as glycerol, m-cresol and polysorbate (Tween®) byaddition of concentrated solutions of these excipients within themixture. If necessary, the pH is adjusted to 7.

Part A

AA: Synthesis of the Hydrophobic Molecules in which p=1

The hydrophobic radicals are represented in the following table by thecorresponding hydrophobic molecule before grafting onto the co-polyaminoacid.

TABLE 1A list and structures of the hydrophobic molecules synthesizedaccording to the invention. No. Structure of the hydrophobic moleculebefore grafting onto the co-polyamino acid AA1

AA2

AA3

AA4

AA5

AA6

AA7

AA8

AA9

AA10

AA12

AA14

EXAMPLE AA1: MOLECULE AA1 Molecule A1: Product Obtained by the ReactionBetween Palmitoyl Chloride and L-Proline.

A solution of palmitoyl chloride (23.0 g, 83.7 mmol) in acetone (167 mL)is added dropwise within 90 minutes to a solution of L-proline (10.6 g,92.1 mmol) in 1 N aqueous sodium hydroxide (230 mL; 230 mmol). After 14h of stirring at room temperature, the heterogeneous mixture is cooledto 0° C., then filtered through a sintered filter to yield a white solidwhich is washed with water (2×100 mL), then diisopropyl ether (100 mL).The solid is dried at reduced pressure. The solid is then dissolved atreflux in 200 mL of water, then 8 mL of 37% hydrochloric acid solutionare added until obtaining pH=1. The opalescent reaction mixture is thencooled to 0° C. The precipitate obtained is filtered through a sinteredfilter, then washed with water (5×50 mL) until filtrates ofphysiological pH from 6.0 to 8.0 are obtained, to be dried subsequentlyin an oven at 50° C. under a vacuum overnight. The product is purifiedby recrystallization in diisopropyl ether. A white solid is obtained.

Yield: 22.7 g (77%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.19-1.45 (24H); 1.58-1.74 (2H);1.88-2.14 (3H); 2.15-2.54 (3H); 3.47 (1H); 3.58 (1H); 4.41 (0.1H); 4.61(0.9H) 6.60-8.60 (1H).

Molecule A2: Product Obtained by Reaction Between Molecule A1 andBoc-Ethylenediamine.

N,N-diisopropylethylamine (DIPEA) (68.8 g, 532.3 mmol),1-hydroxybenzotriaolze (HOBt) (37.1 g, 274.6 mmol), thenN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (53.1 g, 277.0mmol) are added successively and at room temperature to a solution ofmolecule A1 (75.1 g, 212.4 mmol) in 1500 mL of chloroform. After 15minutes of stirring at room temperature, a solution ofBoc-ethylenediamine (Boc-ethylenediamine) (37.6 g, 234.7 mmol) in 35 mLof chloroform is added. After 18 h of stirring at room temperature, a0.1 N HCl solution (2.1 L), then a saturated NaCl solution (1 L) areadded. The phases are separated, then the organic phase is washedsuccessively with a 0.1 N HCl/saturated NaCl solution (2.1 L/1 L), asaturated NaCl solution (2 L), a saturated NaHCO₃ solution (2 L), then asaturated NaCl solution (2 L). The organic phase is dried over anhydroussodium sulfate, filtered, then concentrated at reduced pressure. Thesolid obtained is purified by triturations in diisopropyl ether (3×400mL), to yield a solid after drying under a vacuum at 40° C.

Yield: 90.4 g (86%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.20-1.37 (24H); 1.44 (9H); 1.54-1.70(2H); 1.79-1.92 (1H); 1.92-2.04 (1H); 2.03-2.17 (1H); 2.17-2.44 (3H);3.14-3.36 (4H); 3.43 (1H); 3.56 (1H); 4.29 (0.1 H); 4.51 (0.9 H); 4.82(0.1H); 5.02 (0.9H); 6.84 (0.1H); 7.22 (0.9H).

Molecule AA1

A 4 M hydrochloric acid solution in dioxane (100 mL, 400 mmol) is addeddropwise at 0° C. to a solution of molecule A2 (20.1 g, 40.5 mmol) in330 mL of dichloromethane. After 3 h 30 of stirring at room temperature,the solution is concentrated at reduced pressure. The residue ispurified by flash chromatography (methanol, dichloromethane) to yield awhite solid of molecule AA1 in hydrochloride salt form.

Yield: 16.3 g (93%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.07-1.40 (24H); 1.49-1.63 (2H);1.77-2.18 (4H); 2.18-2.45 (2H); 3.14-3.32 (2H); 3.42-3.63 (2H);3.63-3.84 (2H); 4.37 (0.1H); 4.48 (0.9H); 6.81-8.81 (4H).

LC/MS (ESI): 396.5; (calculated ([M+H]⁺): 396.4).

EXAMPLE AA2: MOLECULE AA2

Molecule A3: 15-methylhexadecan-1-ol.

Magnesium (9.46 g, 389 mmol) in the form of chips is introduced into athree-neck flask under argon. The magnesium is covered with anhydrousTHF (40 mL), and a few drops of 1-bromo-3-methylbutane are added at roomtemperature to initiate the reaction. After the observation of anexothermic reaction and slight turbidity of the medium, the rest of the1-bromo-3-methylbutane (53.87 g, 357 mmol) is added dropwise within 90min, while the temperature of the medium remains stable from 50 to 60°C. The reaction medium is then heated at 70° C. for 2 h.

A solution of 12-bromo-1-dodecanol (43 g, 162.1 mmol) in THF (60 mL) isadded dropwise to a solution of CuCl (482 mg, 4.86 mmol) dissolved inNMP (62 mL) at 0° C. in a three-neck flask are argon. The solution ofextemporaneously prepared hot organic magnesium solution is then addeddropwise to this solution in a manner so as to maintain the temperatureof the medium below 20° C. The mixture is then stirred at roomtemperature for 16 h. The medium is cooled to 0° C. and the reaction isstopped by addition of a 1N aqueous HCl solution until the pH is 1, andthe medium is extracted with ethyl acetate. After washing of the organicphase with a saturated NaCl solution and drying over Na₂SO₄, thesolution is filtered and concentrated under a vacuum to yield an oil.After purification by DCVC on a silica gel (cyclohexane, ethyl acetate),an oil which crystallizes at room temperature is obtained.

Yield: 32.8 g (74%)

¹H NMR (CDCl₃, ppm): 0.87 (6H); 1.14 (2H); 1.20-1.35 (22H); 1.50-1.55(3H); 3.64 (2H).

Molecule A4: 15-methylhexadecanoic acid.

Potassium permanganate (38.2 g, 241.5 mmol) is added in small portionsto a solution of molecule A3 (20.65 g, 80.5 mmol) and tetrabutylammoniumbromide (14.02 g, 42.5 mmol) in a mixture of aceticacid/dichloroethane/water (124/400/320 mL) at room temperature. Afterstirring at reflux for 5 h and return to room temperature, the medium isacidified at pH 1 by gradual addition of 5N HCl. Na₂SO₃ (44.6 g, 354.3mmol) is then added gradually until discoloration of the medium. Theaqueous phase is extracted with dichloromethane, and the combinedorganic phases are dried over Na₂SO₄, filtered and concentrated under avacuum. After purification by chromatography on silica gel (cyclohexane,ethyl acetate, acetic acid), a white solid is obtained.

Yield: 19.1 g (quantitative)

¹H NMR (CDCl₃, ppm): 0.87 (6H); 1.14 (2H); 1.22-1.38 (20H); 1.51 (1H);1.63 (2H); 2.35 (2H).

Molecule A5: Product Obtained by Reaction Between Molecule A4 andL-Proline.

Dicyclohexyl carbodiimide (DCC) (8.01 g, 38.8 mmol) andN-hydroxysuccinimide (NHS) (4.47 g, 38.8 mmol) are added successively toa solution of molecule A4 (10 g, 37 mmol) in THF (360 mL) at 0° C. After17 h of stirring at room temperature, the medium is cooled to 0° C. for20 min, filtered through a sintered filter. L-Proline (4 g, 37.7 mmol),trimethylamine (34 mL) and water (30 mL) are added to the filtrate.After stirring at room temperature for 20 h, the medium is treated witha 1N aqueous HCl solution until the pH is 1. The aqueous phase isextracted with dichloromethane (2×125 ml). The combined organic phasesare washed with a 1N aqueous HCl solution (2×100 ml), water (100 mL),then a saturated aqueous NaCl solution (100 mL). After drying overNa₂SO₄, the organic phase is filtered, concentrated under a vacuum, andthe residue is purified by chromatography on silica gel (cyclohexane,ethyl acetate, acetic acid).

Yield: 9.2 g (72%)

¹H NMR (CDCl₃, ppm): 0.86 (6H); 1.14 (2H); 1.22-1.38 (20H); 1.50 (1H);1.67 (2H); 1.95-2.10 (3H); 2.34 (2H); 2.49 (1H); 3.47 (1H); 3.56 (1H);4.61 (1H).

LC/MS (ESI): 368.3; (calculated ([M+H]⁺): 368.6).

Molecule A6: Product Obtained by Reaction Between Molecule A5 andBoc-Ethylenediamine.

Triethylamine (TEA) (5.23 mL) and2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) at room temperature are added to a solution of molecule A5 (9.22g, 25.08 mmol) in a mixture of THF/DMF (200/50 mL). After 10 min ofstirring, Boc-ethylenediamine (4.42 g, 27.6 mmol) is added. Afterstirring at room temperature for 17 h, the mixture is diluted with water(300 mL) at 0° C. and cold stirred for 20 min. The precipitate formed isfiltered through a sintered filter, and the filtrate is extracted withethyl acetate. The combined organic phases are washed with a saturatedsolution of NaHCO₃, washed over Na₂SO₄, filtered, concentrated under avacuum, and the residue is purified by flash chromatography (ethylacetate, methanol).

Yield: 6.9 g (54%)

¹H NMR (CDCl₃, ppm): 0.86 (6H); 1.15 (2H); 1.22-1.38 (20H); 1.43 (9H);1.50 (1H); 1.64 (4H); 1.85 (1H); 1.95 (1H); 2.10 (1H); 2.31 (2H);3.20-3.35 (3H); 3.45 (1H); 3.56 (1H); 4.51 (In); 5.05 (1H); 7.24 (1H).

LC/MS (ESI): 510.6; (calculated ([M+H]⁺): 510.8).

Molecule AA2

A 4N HCl solution in dioxane (13 mL) is added to a solution of moleculeA6 (5.3 g, 10.40 mmol) in dichloromethane (50 mL) at 0° C. After 5 h ofstirring at 0° C., the medium is concentrated under a vacuum, taken upin water and lyophilized to yield a white solid of molecule AA2 inhydrochloride salt form.

Yield: 4.6 g (99%)

¹H NMR (D₂O, ppm): 0.91 (6H); 1.22 (2H); 1.22-1.50 (20H); 1.63 (3H);1.98 (1H); 2.10 (2H); 2.26 (1H); 2.39 (1H); 2.43 (1H); 3.22 (2H);3.45-3.60 (3H); 3.78 (1H); 4.42 (1H).

LC/MS (ESI): 410.4; (calculated ([M+H]⁺): 410.7).

EXAMPLE AA3: MOLECULE AA3

Molecule A7: Product Obtained by the Reaction Between Molecule A1 andBoc-tri(ethylene glycol)diamine.

By a method similar to the one used for the preparation of molecule A2,applied to molecule A1 (4.0 g, 11.3 mmol) and to Boc-tri(ethyleneglycol)diamine (3.1 g, 12.4 mmol), a colorless oil is obtained afterpurification by flash chromatography (methanol, toluene).

Yield: 5.5 g (84%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.09-1.39 (24H); 1.44 (9H); 1.64 (2H);1.79-2.01 (2H); 2.06-2.43 (4H); 3.23-3.68 (14H); 4.33 (0.2H); 4.56(0.8H); 5.25 (1H); 6.49 (0.2H); 7.13-7.50 (0.8H).

Molecule AA3

By a method similar to the one used for the preparation of molecule AA1,applied to molecule A7 (5.5 g, 9.4 mmol), a white solid of molecule AA3in hydrochloride salt form is obtained after purification by flashchromatography (methanol, dichloromethane).

Yield: 4.3 g (92%).

¹H NMR (DMSO-d6, ppm): 0.85 (3H); 1.08-1.40 (24H); 1.40-1.52 (2H);1.71-2.02 (4H); 2.02-2.31 (2H); 2.90-2.98 (2H); 3.15-3.47 (5H);3.50-3.66 (7H); 4.24 (0.6H); 4.32 (0.4H); 7.83 (0.6H); 7.95 (3H); 8.17(0.4H).

LC/MS (ESI): 484.6; (calculated ([M+H]⁺): 484.4).

EXAMPLE AA4: MOLECULE AA4

Molecule A8: Product Obtained by the Reaction Between Molecule A1 andBoc-1-amino-4,7,10-trioxa-13-tridecaneamine.

By a method similar to the one used for the preparation of molecule A2,applied to molecule A1 (4.5 g, 12.7 mmol) and toBoc-1-amino-4,7,10-trioxa-13-tridecaneamine (4.5 g, 14.0 mmol), a yellowoil is obtained after purification by flash chromatography (methanol,dichloromethane).

Yield: 7.7 g (92%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.22-1.37 (2H); 1.44 (9H); 1.59-1.67(2H); 1.67-2.00 (6H); 2.06-2.45 (4H); 3.18-3.76 (18H); 4.28 (0.2H); 4.52(0.8H); 4.69-5.04 (1H); 6.77 (0.2H); 7.20 (0.8H).

Molecule AA4

By a method similar to the one used for the preparation of molecule AA1,applied to molecule A5 (7.7 g, 11.8 mmol), a yellow oil is obtainedafter purification by flash chromatography (methanol, dichloromethane).Coevaporation with diisopropyl ether makes it possible to obtain themolecule AA4 in hydrochloride salt form in the form of a white solidwhich is dried under a vacuum at 50° C.

Yield: 5.4 g (76%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.08-1.40 (24H); 1.49-1.65 (2H);1.76-2.39 (10H); 3.07-3.28 (3H); 3.34-3.80 (15H); 4.34 (0.05H); 4.64(0.95H); 7.35 (0.05H); 7.66-8.58 (3.95H).

LC/MS (ESI): 556.7; (calculated ([M+H]⁺): 556.5).

EXAMPLE AA5: MOLECULE AA5

Molecule A9: Product Obtained by Reaction Between Molecule A1 andN-Boc-L-lysine methyl ester.

By a method similar to the one used for the preparation of molecule A2,applied to molecule A1 (4 g, 11.3 mmol) and to N-Boc-L-lysine methylester (3.2 g, 12.4 mmol), a colorless oil is obtained after purificationby flash chromatography (methanol, dichloromethane).

Yield: 4.9 g (73%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 0.99-1.54 (37H); 1.54-1.75 (3H);1.75-2.04 (3H); 2.04-2.41 (4H); 2.94-3.19 (2H); 3.19-3.81 (5H);4.28-4.64 (2H); 4.94 (1H); 6.45 (0.1H); 7.36 (0.9H).

LC/MS (ESI): 596.7; (calculated ([M+H]⁺): 596.5).

Molecule A10: Product Obtained by Treatment of Molecule A9 with Ammonia.

320 mL of a 7N ammonia solution in methanol are added to a suspension ofmolecule A9 (4.9 g, 8.2 mmol) in 10 mL of methanol. After 19 h ofstirring at room temperature in closed atmosphere, an additional 100 mLof ammonia solution are added. After 24 h of stirring at roomtemperature in closed atmosphere, the reaction medium is concentrated atreduced pressure. The residue is purified by trituration in diisopropylether at reflux (100 mL) to yield a white solid which is dried under avacuum at 50° C.

Yield: 4.1 g (85%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.06-1.57 (37H); 1.57-1.79 (3H);1.88-2.41 (7H); 3.09 (2H); 3.49 (1H); 3.62 (1H); 4.34 (1H); 4.51 (1H);4.69-4.81 (1H); 5.43 (0.95H); 5.57 (0.05H); 6.25 (0.05H); 6.52 (0.95H);6.83 (0.05H); 7.11 (0.95H).

Molecule AA5

By a method similar to the one used for the preparation of molecule AA1,applied to molecule A10 (388 mg, 0.67 mmol), a white solid of moleculeAA5 in hydrochloride salt form is obtained after purification bytrituration in diisopropyl ether.

Yield: 292 mg (85%).

¹H NMR (DMSO-d6, ppm): 0.85 (3H); 1.06-2.34 (38H); 2.61-2.81 (2H);3.29-3.68 (21H); 4.05-4.17 (1.7H); 4.42 (0.3H); 7.00 (1H); 7.16 (0.7H);7.43 (0.3H); 7.73-8.04 (3.7H); 8.16 (0.3H).

LC/MS (ESI): 481.6; (calculated ([M+H]⁺): 481.4).

EXAMPLE AA6: MOLECULE AA6 Molecule A11: Product Obtained by the ReactionBetween Stearoyl Chloride and L-proline.

By a method similar to the one used for the preparation of molecule A1,applied to L-proline (5.0 g, 43.4 mmol) and to stearoyl chloride (12.0g, 39.6 mmol), a white solid is obtained after purification by flashchromatography (methanol, dichloromethane).

Yield: 5.37 g (36%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.26-1.37 (28H); 1.64-1.70 (2H);1.88-2.10 (3H); 2.36 (2H); 2.54-2.58 (1H).; 3.46 (1H); 3.56 (1H); 4.62(1H).

LC/MS (ESI): 382.6; (calculated ([M+H]⁺): 382.3).

Molecule A12: Product Obtained by Reaction Between Molecule A11 andBoc-tri(ethyleneglycol)diamine.

By a method similar to the one used for the preparation of molecule A6,applied to molecule A11 (33.81 g, 88.6 mmol) and to Boc-tri(ethyleneglycol)diamine (26.4 g, 106.3 mmol) in THF using DIPEA instead of TEA, awhite solid is obtained after purification by flash chromatography(ethyl acetate, methanol).

Yield: 43.3 g (80%)

¹H NMR (CDCl₃, ppm): 0.87 (3H); 1.24 (30H); 1.43 (9H); 1.61 (2H); 1.82(1H); 1.96 (1H); 2.25-2.45 (2H); 3.25-3.65 (14H); 4.30 (0.15H); 4.53(0.85H); 5.25 (1H); 6.43 (0.15H); 7.25 (0.85H).

LC/MS (ESI): 612.6; (calculated ([M+H]⁺): 612.9).

Molecule AA6

By a method similar to the one used for the preparation of molecule AA2,applied to molecule A12 (43 g, 70.3 mmol), the residue obtained afterconcentration under a vacuum is triturated in acetonitrile. Thesuspension is filtered, and the solid is washed with acetonitrile, thenacetone. After drying under a vacuum, a white solid of molecule AA6 inhydrochloride salt form is obtained.

Yield: 31.2 g (81%)

¹H NMR (DMSO-d₆, ppm): 0.85 (3H); 1.23 (28H); 1.45 (2H); 1.70-2.05 (4H);2.13 (1H); 2.24 (1H); 2.95 (2H); 3.10-3.25 (2H); 3.30-3.65 (10H);4.20-4.45 (1H); 7.85-8.25 (4H).

LC/MS (ESI): 512.4; (calculated ([M+H]⁺): 512.8).

EXAMPLE AA7: MOLECULE AA7 Molecule A13: Product Obtained by ReactionBetween Arachidonic Acid and L-proline.

By a method similar to the one used for the preparation of molecule A5,applied to arachidonic acid (15.51 g, 49.63 mmol) and to L-proline (6 g,52.11 mmol) using DIPEA instead of TEA, a white solid is obtained afterpurification by column chromatography on silica gel (cyclohexane, ethylacetate, acetic acid).

Yield: 12.9 g (63%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.28 (34H); 1.66 (2H); 1.95-2.15 (2H);2.34 (2H); 2.45 (1H); 3.47 (1H); 3.56 (1H); 4.60 (1H).

LC/MS (ESI): 410.4; (calculated ([M+H]⁺): 410.6).

Molecule A14: Product Obtained by the Reaction Between Molecule A13 andBoc-1-amino-4,7,10-trioxa-13-tridecane.

By a method similar to the one used for the preparation of molecule A12,applied to molecule A13 (10.96 g, 26.75 mmol) and toBoc-1-amino-4,7,10-trioxa-13-tridecane (10.29 g, 32.11 mmol), a solid isobtained after purification by column chromatography on silica gel(cyclohexane, ethyl acetate, methanol).

Yield: 14.2 g (75%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.24 (32H); 1.43 (9H); 1.57-2.00 (8H);2.10-2.45 (4H); 3.20-3.75 (18H); 4.30 (0.20H); 4.55 (0.80H); 5.03 (1H);6.75 (0.20H); 7.20 (0.80H).

LC/MS (ESI): 712.8; (calculated ([M+H]⁺): 713.1).

Molecule AA7

After a method similar to the one used for the preparation of moleculeAA2, applied to molecule A14 (14.25 g, 20.01 mmol), the residue obtainedafter concentration under a vacuum of the reaction medium is dissolvedin methanol and evaporated at reduced pressure, the operation beingrepeated 4 times to yield a white solid of molecule AA7 in hydrochloridesalt form.

Yield: 12.7 g (98%)

¹H NMR (DMSO-d₆, ppm): 0.85 (3H); 1.23 (32H); 1.45 (2H); 1.64 (2H);1.70-2.05 (6H); 2.10-2.30 (2H); 2.82 (2H); 3.08 (2H); 3.30-3.60 (14H);4.15-4.30 (1H); 7.73-8.13 (4H).

LC/MS (ESI): 612.7; (calculated ([M+H]⁺): 612.9).

EXAMPLE AA8: MOLECULE AA8

Molecule A15: Product Obtained by the Reaction Between L-leucine andpalmitoyl chloride.

By a method similar to the one used for the preparation of molecule A1,applied to L-leucine (15.0 g, 114.4 mmol) and to palmitoyl chloride(34.5 g, 125 mmol), a white solid is obtained by trituration indiisopropyl ether.

Yield: 13.0 g (31%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 0.96 (6H); 1.16-1.35 (24H); 1.55-1.77(5H); 2.23 (2H); 4.55-4.60 (1H); 5.88 (1H).

Molecule A6: Product Obtained by the Reaction Between Molecule A15 andL-proline methyl ester

By a method similar to the one used for the preparation of molecule A2,applied to molecule A15 (6.00 g, 16.2 mmol) and to L-proline methylester (3.23 g, 19.5 mmol), a slightly yellow oil is obtained afterpurification by flash chromatography (methanol, dichloromethane).

Yield: 5.8 g (74%)

¹H NMR (CDCl₃, ppm): 0.83-1.00 (9H); 1.18-1.32 (24H); 1.40-1.73 (5H);1.84-2.33 (6H); 3.47-3.89 (2H); 3.70 (1.14H); 3.71 (1.21H); 3.74(0.53H); 3.76 (0.12H); 4.40-4.56 (1H); 4.63-4.67 (0.04H); 4.84 (0.38);4.90 (0.40); 5.06 (0.18); 5.99 (0.18H); 6.08-6.21 (0.82).

LC/MS (ESI): 481.6; (calculated ([M+H]⁺): 481.4).

Molecule a 17: Product Obtained by Saponification of the Methyl Ester ofMolecule A16.

1N sodium hydroxide (13.5 mL, 13.5 mmol) is added to a solution ofmolecule A16 (5.8 g, 12.06 mmol) in 30 mL of methanol. After 20 h ofstirring at room temperature, the solution is diluted with water, thenacidified with 20 mL of 1 N hydrochloric acid at 0° C. The precipitateis filtered, then rinsed with water (50 mL), before it being solubilizedin 50 mL of dichloromethane. The organic phase is dried over Na₂SO₄,filtered, then concentrated at reduced pressure to yield a colorlessoil.

Yield: 4.5 g (80%)

¹H NMR (CDCl₃, ppm): 0.85-0.99 (9H); 1.14-1.41 (2414); 1.43-1.72 (5H);1.87-2.47 (7H); 3.48-3.55 (0.6H); 3.56-3.62 (0.41H); 3.83-3.90 (0.4H);3.90-3.96 (0.6H); 4.52-4.56 (0.6H); 4.56-4.59 (0.41H); 4.80-4.86 (0.4H);4.86-4.91 (0.6H); 6.05 (0.4H); 6.11 (0.6H).

LC/MS (ESI): 467.6; (calculated ([M+H]⁺): 467.4).

Molecule A18: Product Obtained by the Reaction BetweenBoc-ethylenediamine and Molecule A17.

By a method similar to the one used for the preparation of molecule A2,applied to molecule A17 (4.5 g, 9.64 mmol) and to Boc-ethylenediamine(1.70 g, 10.61 mmol), a colorless oil is obtained after purification byflash chromatography (methanol, dichloromethane).

Yield: 2.0 g (34%)

¹H NMR (CDCl₃, ppm): 0.83-0.99 (9H); 1.19-1.32 (24H); 1.44 (9H);1.48-2.37 (14H); 3.09-3.99 (4H); 4.28-5.01 (2H); 5.64-6.04 (1H);6.87-7.06 (1H).

LC/MS (ESI): 609.7; (calculated ([M+H]⁺): 609.5).

Molecule AA8

By a method similar to the one used for the preparation of molecule AA1,applied to molecule A18 (2 g, 3.28 mmol), a solid of molecule AA8 inhydrochloride salt form is obtained after purification by flashchromatography (methanol, dichloromethane).

Yield: 1.5 g (90%)

¹H NMR (CDCl₃, ppm): 0.83-1.00 (9H); 1.18-1.32 (24H); 1.37-1.77 (5H);1.93-2.41 (6H); 3.07-3.97 (6H); 4.44-4.77 (2H); 7.66-8.21 (2H).

LC/MS (ESI): 509.6; (calculated ([M+H]⁺): 509.4).

EXAMPLE AA9: MOLECULE AA9 Molecule A19: Product Obtained by the ReactionBetween Lauric Acid and L-phenylalanine.

By a method similar to the one used for the preparation of molecule A5,applied to lauric acid (8.10 g, 40.45 mmol) and to L-phenylalanine (7 g,42.38 mmol), a white solid is obtained.

Yield: 12.7 g (98%)

¹H NMR (DMSO-d₆, ppm): 0.86 (3H); 1.10-1.30 (16H); 1.36 (2H); 2.02 (2H);2.82 (1H); 3.05 (1H); 4.42 (1H); 7.15-7.30 (5H); 8.05 (1H); 12.61 (1H).

LC/MS (ESI): 348.2; (calculated ([M+H]⁺): 348.5).

Molecule A20: Product Obtained by the Reaction Between Molecule A19 andthe Hydrochloride Salt of the Methyl Ester of L-proline.

By a method similar to the one used for the preparation of molecule A6,applied to molecule A19 (9.98 g, 28.72 mmol) and to the hydrochloridesalt of the methyl ester of L-proline (5.23 g, 31.59 mmol), a colorlessoil is obtained after purification by column chromatography on silicagel (cyclohexane, ethyl acetate).

Yield: 5.75 g (44%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.10-1.30 (16H); 1.50-1.75 (3H);1.80-2.02 (3H); 2.17 (2H); 2.65 (0.5H); 2.95 (1K); 3.05-3.20 (1.5H);3.50-3.65 (1H); 3.75 (3H); 4.29 (0.5H); 4.46 (0.5H); 4.70 (0.1H); 4.95(0.9H); 6.20-6.30 (1H); 7.15-7.30 (5H).

LC/MS (ESI): 459.2; (calculated ([M+H]⁺): 459.6).

Molecule A21: Product Obtained by Saponification of Molecule A20.

Lithium hydroxide (LiOH) (600.49 mg, 25.07 mmol) is added to a solutionof molecule A20 (5.75 g, 12.54 mmol) in a mixture of THF/methanol/water(40/40/40 mL) at 0° C., then the mixture is stirred at room temperaturefor 20 h. After evaporation of the organic solvents in a vacuum, theaqueous phase is diluted in water, acidified with a 1 N aqueous HClsolution until the pH is 1. The product is then extracted with ethylacetate. The combined organic phases are washed with a saturated aqueousNaCl solution, dried over Na₂SO₄, filtered and concentrated at reducedpressure to yield a colorless oil.

Yield: 5.7 g (quantitative)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.10-1.30 (16H); 1.50-1.80 (3H);1.67-2.02 (2H); 2.20 (2H); 2.25 (0.4H); 2.60 (0.6H); 2.85-3.10 (2.6H);3.55-3.65 (1.4H); 4.35 (0.6H); 4.55 (0.4H); 4.94 (1H); 6.28 (0.4H); 6.38(0.6H); 7.20-7.30 (51H).

LC/MS (ESI): 445.2; (calculated ([M+H]⁺): 445.6).

Molecule A22: Product Obtained by Reaction Between Boc-ethylenediamineand Molecule A21.

By a method similar to the one used for the preparation of molecule A6,applied to molecule A21 (5.67 g, 12.75 mmol) and to Boc-ethylenediamine(2.25 g, 14.03 mmol), a colorless oil is obtained after purification bycolumn chromatography on silica gel (dichloromethane, methanol).

Yield: 5.7 g (76%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.25 (16H); 1.43 (9H); 1.58 (2.6H);1.75-1.95 (1.4H); 2.15-2.30 (3H); 2.64 (0.5H); 2.95-3.10 (2.5H);3.20-3.40 (4H); 3.45 (0.5H); 3.55 (0.2H); 3.66 (1H); 4.44 (1H); 4.50(0.2H); 4.60 (0.6H); 4.99 (0.7H); 5.54 (0.5H); 5.95 (0.2H); 6.17 (1H);6.60 (0.5H); 7.07 (0.5H); 7.20-7.40 (5H).

LC/MS (ESI): 587.4; (calculated ([M+H]⁺): 587.8).

Molecule AA9

After a method similar to the one used for the preparation of moleculeAA2, applied to molecule A22 (5.66 g, 9.65 mmol), the residue obtainedafter concentration under a vacuum of the reaction medium is dissolvedin methanol and evaporated at reduced pressure, the operation beingrepeated 4 times to yield a white foam of molecule AA9 in hydrochloridesalt form.

Yield: 4.9 g (97%)

¹H NMR (DMSO-d₆, 120° C., ppm): 0.89 (3H); 1.26 (16H); 1.43 (2H); 1.68(0.6H); 1.75-2.00 (3H); 2.05-2.25 (2.4H); 2.82-3.05 (5H); 3.38 (2H);3.50-3.70 (1.4H); 4.25 (0.6H); 4.63 (0.4H); 4.77 (0.6H); 7.25-7.50 (5H);7.55-8.20 (4H).

LC/MS (ESI): 487.4; (calculated ([M+H]⁺): 487.7).

EXAMPLE AA10: MOLECULE AA10 Molecule A23: Product Obtained by theReaction Between Nipecotic Acid and Arachidonic Acid

By a method similar to the one used for the preparation of molecule A5,applied to arachidonic acid (2.30 g, 7.37 mmol) and to nipecotic acid(1.00 g, 7.74 mmol), a white solid is obtained after filtration of theaqueous phase acidified until the pH is 1, and washing of the solid withwater, then dichloromethane.

Yield: 1.65 g (53%)

1H NMR (CDCl₃, ppm): 0.88 (3H); 1.07-1.88 (37H); 2.10 (1H); 2.28-2.45(2H); 2.52 (1H); 2.91-3.17 (1.5H); 3.42 (0.5H); 3.72 (0.5H); 3.84(0.5H); 4.08 (0.5H); 4.56 (0.5H).

LC/MS (ESI): 424.4; 848.0; (calculated ([M+H]⁺): 424.4; ([2M+H]⁺):847.8).

Molecule A24: Product Obtained by the Reaction Between Molecule A23 andBoc-1-amino-4,7,10-trioxa-13-tridecaneamine.

DIPEA (1.01 g, 7.79 mmol) and TBTU (1.31 g, 4.09 mmol) are addedsuccessively and at room temperature to a suspension of molecule A23(1.65 g, 3.89 mmol) in 20 mL of THF. After 30 minutes of stirring,Boc-1-amino-4,7,10-trioxa-13-tridecaneamine (1.37 g, 4.28 mmol) isadded, and the reaction medium is stirred at room temperature for 18 h.After evaporation of the solvent at reduced pressure, the residue isdiluted with ethyl acetate (100 mL), the organic phase is washedsuccessively with a saturated aqueous NaHCO₃ solution, a 1 N aqueous HClsolution, a saturated aqueous NaCl solution, dried over Na₂SO₄, filteredand concentrated at reduced pressure. A white solid is obtained afterpurification by flash chromatography (cyclohexane, ethyl acetate,methanol).

Yield: 1.97 g (70%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.15-2.70 (54H); 3.10-3.46 (6H);3.46-3.71 (12.6H); 3.92 (0.4H); 4.17 (0.6H); 4.49 (0.4H); 4.80-5.16(1H); 6.35-6.76 (1H).

LC/MS (ESI): 726.8; (calculated ([M+H]⁺: 726.6).

Molecule AA10

By a method similar to the one used for the preparation of molecule AA1,applied to molecule A24 (1.97 g, 2.71 mmol), a white solid of moleculeAA10 is obtained after evaporation of the solvent, trituration inacetone, filtration and washing with acetone, then drying at reducedpressure at 50° C.

Yield: 1.66 g (92%)

¹H NMR (DMSO-d₆, ppm): 0.86 (3H); 1.09-1.90 (42H); 2.05-2.68 (5H);2.45-2.68 (1H); 2.78-3.19 (6H); 3.36-3.44 (2H); 3.44-3.60 (10H);3.69-3.87 (1H); 4.20 (0.4H); 4.35 (0.6H).

LC/MS (ESI): 626.7; (calculated ([M+H]⁺): 626.5).

EXAMPLE AA12: MOLECULE AA12 Molecule A26: Product Obtained by theReaction Between Myristoyl Chloride and L-proline

Myristoyl chloride (322 g, 1.30 mol) in solution in dichloromethane(1.63 L) is added slowly within 1 h to a solution of L-proline (300.40g, 2.61 mol) in 2 N aqueous sodium hydroxide (1.63 L) at 0° C. At theend of the addition, the temperature of the reaction medium is broughtback to 20° C. within 2 h, then stirred for an additional 2 h. Themixture is cooled to 0° C., then a 37% HCl solution (215 mL) is addedwithin 15 minutes. The reaction medium is stirred for 10 min at 0° C.,then for 1 h from 0° C. to 20° C. The organic phase is separated, washedwith a 10% HCl solution (3×430 mL), a saturated aqueous NaCl solution(430 mL), dried over Na₂SO₄, filtered through cotton, then concentratedat reduced pressure. The residue is solubilized in heptane (315 mL),then pentane (1.6 L) is added under mechanical stirring. A white solidis obtained after filtration through a sintered filter and drying atreduced pressure.

Yield: 410.6 g (97%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.28 (20H); 1.70 (2H); 1.90-2.10 (3H);2.36 (2H); 2.51 (1H); 3.47 (1H); 3.56 (1H); 4.61 (1H).

LC/MS (ESI): 326.4; 651.7; (calculated ([M+H]⁺): 326.3; ([2M+H]⁺):651.6).

Molecule A27: Product Obtained by the Reaction Between Molecule A26 andBoc-ethylenediamine.

HOBt (1.83 g, 11.98 mmol), then Boc-ethylenediamine (1.62 g, 10.14 mmol)are added successively to a solution of molecule A26 (3.00 g, 9.21 mmol)at room temperature in methyl-THF (50 mL), and the medium is cooled to0° C. EDC (2.29 g, 11.98 mmol) is added, then the mixture is stirred for17 h between 0° C. and room temperature. The reaction medium is thenwashed with a saturated aqueous NH₄Cl solution (50 mL), a saturatedaqueous NaHCO₃ solution (50 mL), then a saturated aqueous NaCl solution(50 mL), dried over Na₂SO₄, filtered and concentrated at reducedpressure. A white solid is obtained after recrystallization in methanol.

Yield: 2.34 g (49%).

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.16-1.38 (20H); 1.44 (9H); 1.56-1.71(2H); 1.78-2.45 (6H); 3.11-3.72 (6H); 4.30 (0.1H); 4.51 (0.9H); 4.87(0.1H); 5.04 (0.9H); 6.87 (0.1H); 7.23 (0.9H).

LC/MS (ESI): 468.0; (calculated ([M+H]⁺): 468.4).

Molecule AA12

By a method similar to the one used for the preparation of molecule AA1,applied to molecule A27 (2.34 g, 5.00 mmol), a white solid of moleculeAA12 is obtained after evaporation of the solvent and triturations indiisopropyl ether.

Yield: 1.5 g (74%)

¹H NMR (MeOD-d4, ppm): 0.90 (3H); 1.21-1.43 (20H); 1.54-1.66 (2H);1.85-2.28 (4H); 2.39 (2H); 3.00-3.17 (2H); 3.30-3.40 (1H); 3.43-3.71(3H); 4.29 (0.94H); 4.48 (0.06H).

LC/MS (ESI): 368.2; (calculated ([M+H]⁺): 368.3).

EXAMPLE AA14: MOLECULE AA14

Resin AA14-1: Product Obtained by the Reaction Between4,7,10-trioxa-1,13-Tridecanediamine and the resin 2-Cl-trityl chloride

DIPEA (8.64 mL, 49.60 mmol) is added to a solution of4,7,10-trioxa-1,13-tridecanediamine (10.87 mL, 49.60 mmol) indichloromethane (50 mL) at room temperature. This solution is pouredonto the resin 2-Cl-trityl chloride which was washed beforehand withdichloromethane (100-200 mesh, 1% DVB, 1.24 mmol/g) (4.00 g, 4.96 mmol)in a reactor adapted for peptide synthesis on a solid support. After 2 hof stirring at room temperature, HPLC grade methanol (0.8 mL/g resin,3.2 mL) is added, and the mixture is stirred at room temperature for 15min. The resin is filtered, washed successively with dichloromethane(3×50 mL), DMF (2×50 mL), dichloromethane (2×50 mL), isopropanol (1×50mL) and dichloromethane (3×50 mL).

Resin AA14-2: Product Obtained by Reaction Between the Resin AA14-1 andFmoc-glycine.

DIPEA (5.18 mL, 29.76 mmol) is added to a suspension of Fmoc-glycine(4.42 g, 14.88 mmol) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide-hexafluorophosphate (HATU, 5.66 g, 14.88 mmol) in a mixture ofDMF/dichloromethane 1:1 (60 mL). After complete solubilization, thesolution obtained is poured onto resin AA14-1. After 2 h of stirring atroom temperature, resin is filtered, washed successively with DMF (3×60mL), isopropanol (1×60 ml) and dichloromethane (3×60 ml).

Resin AA14-3: product obtained by reaction between resin AA14-2 and amixture of DMF/piperidine 80:20. The resin AA14-2 is treated with amixture of DMF/piperidine 80:20 (50 mL). After 30 minutes of stirring atroom temperature, the resin is filtered, washed successively with DMF(3×50 mL), isopropanol (1×50 mL) and dichloromethane (3×50 mL).

Resin AA14-4: Product Obtained by the Reaction Between Resin AA14-3 andFmoc-proline.

By a method similar to the one used for resin AA14-2, applied to resinAA14-3 and Fmoc-proline (5.02 g, 14.88 mmol) in DMF (50 mL), resinAA14-4 is obtained.

Resin AA14-5: Product Obtained by Reaction Between Resin AA14-4 and aMixture of DMF/piperidine 80:20.

By a method similar to the one used for resin AA14-3, applied to resinAA14-4 and a mixture of DMF/piperidine 80:20 (50 mL), resin AA14-5 isobtained.

Resin AA14-6: Product Obtained by Reaction Between Resin AA14-5 andPalmitic Acid.

By a method similar to the one used for the preparation of resin AA14-4,applied to resin AA14-5 and to palmitic acid (3.82 g, 14.88 mmol), resinAA14-6 is obtained.

Molecule AA14

The resin AA14-6 is treated with a mixture of TFA/dichloromethane 1:1(50 mL). After 30 minutes of stirring at room temperature, the resin isfiltered and washed with dichloromethane (3×50 mL). The solvents areevaporated under a vacuum. Two coevaporations are then carried out onthe residue with dichloromethane (50 mL), then diisopropyl ether (50mL). The residue is solubilized in dichloromethane (50 mL), and theorganic phase is washed with a 1 N aqueous NaOH solution (1×50 mL), thena saturated NaCl solution (2×50 mL). After drying over Na₂SO₄, theorganic phase is filtered, concentrated under a vacuum, and the residueis filtered by chromatography on silica gel (dichloromethane, methanol,NH₄OH).

Yield: 1.65 g (54% global over 7 steps)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.18-2.39 (38H); 2.79 (2H); 3.23-3.44(2H); 3.47-3.69 (14H); 3.76 (0.92H); 3.82 (0.08H); 3.98 (0.08H); 4.03(0.92H); 4.34 (0.08H); 4.39 (0.92H); 7.00-7.40 (2H).

LC/MS (ESI): 613.7; (calculated ([M+H]⁺): 613.5).

AB: Synthesis of the co-polyamino acids

TABLE 1b No. co-polyamino acids bearing carboxylates charges andhydrophobic radicals AB1

AB2

AB3

AB4

AB5

AB6

AB7

AB8

AB9

AB10

AB11

AB12

AB13

AB21

AB24

AB25

AB28

Part AB: Synthesis of the Co-Polyamino Acids EXAMPLE AB1: CO-POLYAMINOACID AB1—SODIUM POLY-L-GLUTAMATE MODIFIED BY MOLECULE AA1 AND HAVING ANUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 2900 G/MOL

Co-Polyamino Acid AB1-1:

poly-L-glutamic acid having a relative number average molecular weight(Mn) of 3861 g/mol originating from the polymerization of theγ-benzyl-L-glutamate N-carboxyanhydride initiated by hexylamine.

γ-Benzyl-L-glutamate N-carboxyanhydride (89.9 g, 341 mmol) is placedunder a vacuum for 30 minutes in a round-bottom flask dried in the oven,then anhydrous DMF (200 mL) is introduced. The mixture is then stirredunder argon until the dissolution is complete, cooled to 4° C., thenhexylamine (2.05 mL; 15.5 mmol) is introduced rapidly. The mixture isstirred between 4° C. and room temperature for 2 days. The reactionmixture is then heated at 65° C. for 2 h, cooled at room temperature,then poured dropwise into diisopropyl ether (3 L) under stirring. Thewhite precipitate is recovered by filtration, washed with diisopropylether (2×200 mL), then dried under a vacuum at 30° C. to yield apoly(gamma-benzyl-L-glutamic) acid (BBLG).

Trifluoroacetic acid (TFA, 340 mL) at 4° C., a 33% hydrobromic acidsolution (HBr) in acetic acid (240 mL, 1.37 mol) is added dropwise to asolution of PBLG (74.8 g). The mixture is stirred at room temperaturefor 2 h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropylether and water under stirring (4 L). After 2 h of stirring, theheterogeneous mixture is allowed to rest overnight. The whiteprecipitate is recovered by filtration, washed with a 1:1 (v/v) mixtureof diisopropyl ether and water (340 mL), then with water (340 mL).

The solid obtained is then solubilized in water (1.5 L) by adjusting thepH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a1 N aqueous sodium hydroxide solution. After solubilization, thetheoretical concentration is adjusted to 20 g/L theoretical by additionof water to obtain a final volume 2.1 L.

The solution is filtered through a 0.45-μm filter, then purified byultrafiltration against a 0.9% NaCl solution, then water until theconductimetry of the permeate is less than 50 μS/cm. The solution ofco-polyamino acid is the concentrated until a final volume 1.8 L isobtained.

The aqueous solution is then acidified by addition of 37% hydrochloricacid solution until the pH is 2. After 4 h of stirring, the precipitateobtained is filtered, washed with water (2×340 mL), then dried under avacuum at 30° C. to yield a poly-L-glutamic acid having a number averagemolecular weight (Mn) of 3861 g/mol with respect to a polyoxyethylenestandard (PEG).

Co-Polyamino Acid AB1

The co-polyamino acid AB-1 (10.0 g) is solubilized in DMF (700 mL) at30° C.-40° C., then cooled to 0° C. Molecule AA1 in hydrochloric saltform (1.64 g, 3.8 mmol) is suspended in DMF (23 mL), and triethylamine(0.39 g, 3.8 mmol) is then added and the mixture is heated slightlyunder stirring until the dissolution is complete. To the solution ofco-polyamino acid at 0° C., N-methylmorpholine (NMM, 7.6 g, 75 mmol) inDMF (14 mL) and ethyl chloroformate (ECF, 8.2 g, 75 mmol) are added.After 10 min at 0° C., the solution containing molecule AA1 is added,and the mixture is maintained at 30° C. for 2 h. The reaction mixture ispoured dropwise onto 5.5 L of water containing 15% by weight of sodiumchloride and HCl (pH 2), then it is allowed to rest overnight. Theprecipitate is collected by filtration and dried under a vacuum forapproximately 30 min. The white solid obtained is taken up in water (500mL) and the pH is adjusted to 7 by slow addition of a 1N NaOH aqueoussolution. After filtration through a 0.45-μm filter, the clear solutionobtained is purified by ultrafiltration against a 0.9% NaCl solution,then water until the conductimetry of the permeate is less than 50μS/cm. After discharging, the solution is filtered through a 0.2 μmfilter and stored at 2-8° C.

Dry extract: 24.9 mg/g

An average degree of polymerization (DP) of 23 is estimated by ¹H NMR inD₂O by comparing the integration of the signals originating from thehydrophobe grafted onto that of the signals originating from the mainchain.

Based on ¹H NMR: i=0.05

The calculated average molecular weight of the co-polyamino acid AB1 iscalculated on the basis of the molecular weights of the radicals R₁ andR₂, the aspartate and/or glutamate residue(s) (comprising an amidebond), the hydrophobic radical, the DS and the DP.

The calculated average molecular weight of the co-polyamino acid AB1 is3945 g/mol.

HPLC-aqueous SEC (calibrant PEG): Mn=2900 g/mol.

EXAMPLE AB2: CO-POLYAMINO ACID AB2—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA1 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3700G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB1, applied to the hydrochloride salt of molecule AA1(1.64 g, 3.8 mmol) and to a poly-L-glutamic acid having a relative Mn of5200 g/mol (10.0 g) obtained by a method similar to the one used for thepreparation of the co-polyamino acid AB1-1, a sodium poly-L-glutamatemodified by molecule AA1 is obtained.

Dry extract: 14.1 mg/g

DP (estimated based on ¹H NMR): 35

Based on ¹H NMR: i=0.05

The calculated average molecular weight of the co-polyamino acid AB2 is5972 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3700 g/mol.

EXAMPLE AB3: CO-POLYAMINO ACID AB3—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA1 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4900G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB1, applied to the hydrochloride salt of molecule AA1(3.30 &g, 7.6 mmol) and to a poly-L-glutamic acid having a relativenumber average weight (Mn) of 5200 g/mol (10.0 g) obtained by a methodsimilar to the one used for the preparation of the co-polyamino acidAB1-1, a sodium poly-L-glutamate modified by molecule AA1 is obtained.

Dry extract: 23.4 mg/g

DP (estimated based on ¹H NMR): 35

The calculated average molecular weight of the co-polyamino acid AB3 is6594 g/mol.

Based on ¹H NMR: i=0.10

HPLC aqueous-SEC (calibrant PEG): Mn=4900 g/mol.

EXAMPLE AB4: CO-POLYAMINO ACID AB4—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA2 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 1800G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB1, applied to the hydrochloride salt of molecule AA2(1.09 g, 2.4 mmol) and to a poly-L-glutamic acid having an averageweight Mn=5600 g/mol (6.3 g) obtained by a method similar to the oneused for the preparation of the co-polyamino acid AB1-1 but with a stepof deprotection of the benzyl esters using trimethylsilane iodideaccording to the protocol described in the publication J. Am. Chem. Soc.2000, 122, 26-34 (Subramanian G. et al.), a sodium poly-L-glutamatemodified by molecule AA2 is obtained.

Dry extract: 21.5 mg/g

DP (estimated based on ¹H NMR): 35

Based on ¹H NMR: i=0.052

The calculated average molecular weight of the co-polyamino acid AB4 is6022 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=1800 g/mol.

EXAMPLE AB5: CO-POLYAMINO ACID AB5—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA6 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 2600G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB1, applied to the hydrochloride salt of molecule AA6(2.06 g, 3.8 mmol) and to a poly-L-glutamic acid (9.8 g) obtained by amethod similar to the one used for the preparation of the co-polyaminoacid AB1-1, a sodium poly-L-glutamate modified by molecule AA6 isobtained.

Dry extract: 20.9 mg/g

DP (estimated based on ¹H NMR): 23

Based on ¹H NMR: i=0.05

The calculated average molecular weight of the co-polyamino acid AB5 is4079 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=2600 g/mol.

EXAMPLE AB6: CO-POLYAMINO ACID AB6—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA7 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4000G/MOL

A poly-L-glutamic acid having an average weight Mn=3500 g/mol and adegree of polymerization 22 (10.0 g) obtained by a method similar to theone used for the preparation of the co-polyamino acid AB1-1 issolubilized in DMF (420 mL) at 30° C.-40° C., then maintained at thistemperature. In parallel, the hydrochloride salt of molecule AA7 (1.47g, 2.3 mmol) is suspended in DMF (12 mL), and triethylamine (0.23 g, 2.3mmol) is added, the mixture is then heated slightly under stirring untilthe dissolution is complete. To the solution of co-polyamino acid inDMF, NMM (7.6 g, 75 mmol), the solution of AA7, then 2-hydroxypyridineN-oxide (HOPO, 0.84 g, 7.5 mmol) are added successively. The reactionmixture is then cooled to 0° C., then EDC (1.44 g, 7.5 mmol) is addedand the temperature of the medium is increased to room temperature overa period of 2 h. The reaction medium is filtered through a woven 0.2-mmfilter and poured dropwise onto 3.5 L of water containing 15% by weightof NaCl and HCl (pH 2) under stirring. At the end of the addition, thepH is readjusted to 2 with a 37% HCl solution, and the suspension isallowed to rest overnight. The precipitate is collected by filtration,then rinsed with 100 mL of water. The white solid obtained issolubilized in 500 mL of water by slow addition of a 1 N aqueous NaOHsolution until the pH is 7 under stirring, then the solution is filteredthrough a 0.45-μm filter. The clear solution obtained is purified byultrafiltration against a 0.9% NaCl solution, then water until theconductimetry of the permeate is less than 50 μS/cm. The solution isfiltered through a 0.2-μm filter and stored at 2-8° C.

Dry extract: 21.6 mg/g

DP (estimated based on ¹H NMR): 20

Based on ¹H NMR: i=0.025

The calculated average molecular weight of the co-polyamino acid AB6 is3369 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4000 g/mol.

EXAMPLE AB7: CO-POLYAMINO ACID AB7—SODIUM POLY-L-GLUTAMATE CAPPED AT ONEOF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE AA7 AND HAVING ANUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3300 G/MOL

Co-polyamino acid AB7-1: poly-L-glutamic acid having a relative numberaverage molecular weight (Mn) of 3600 g/mol and DP 21, originating fromthe polymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiatedby hexylamine, and capped at one of its ends by an acetyl group.

γ-Benzyl-L-glutamate N-carboxyanhydride (Glu(OBn)-NCA, 100.0 g, 380mmol) is placed under a vacuum for 30 minutes in a round-bottom flaskpreviously dried in the oven, then anhydrous DMF (225 mL) is introduced.The mixture is then stirred under argon until the dissolution iscomplete, cooled to 4° C., then hexylamine (1.78 g, 17 mmol) isintroduced rapidly. The mixture is stirred between 4° C. and roomtemperature for 2 days, then precipitated in diisopropyl ether (3.4 L).The precipitate is recovered by filtration, washed two times withdiisopropyl ether (225 mL), then dried to yield a white solid which isdissolved in 450 mL of THF. To this solution, DIPEA (31 mL, 176 mmol),then acetic anhydride (17 mL, 176 mmol) are added successively. After anight of stirring at room temperature, the solution is poured slowlyinto diisopropyl ether (3 L) under stirring. After 1 h of stirring, theprecipitate is filtered, washed two times with diisopropyl ether (250mL), then dried under a vacuum at 30° C. to yield apoly(gamma-benzyl-L-glutamic) acid capped at one of its ends by anacetyl group.

A 33% hydrobromic acid solution (HBr) in acetic acid (235 mL) is addeddropwise to a solution of the co-polyamino acid above (72 g) intrifluoroacetic acid (TFA, 335 mL) at 4° C. The mixture is stirred atroom temperature for 3 h 30, then poured dropwise onto a 1:1 (v/v)mixture of diisopropyl ether and water under stirring (4 L). After 2 hof stirring, the heterogeneous mixture is allowed to rest overnight. Thewhite precipitate is recovered by filtration, washed with 1:1 (v/v)mixture of diisopropyl ether and water (340 mL), then with water (340mL.).

The solid obtained is then dissolved in water (1.5 L) by adjusting thepH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a1 N aqueous sodium hydroxide solution. After solubilization, the mixtureis diluted by addition of water to obtain a final volume of 2.1 L. Thesolution is filtered through a 0.45-μm filter, then purified byultrafiltration against a 0.9% NaCl solution, then water until theconductimetry of the permeate is less than 50 μS/cm. The solution ofco-polyamino acid is then concentrated until a final volume of 1.8 L isobtained.

The aqueous solution is then acidified by addition of 37% hydrochloricacid solution until the pH is 2. After 4 h of stirring, the precipitateobtained is filtered, washed with water (330 mL), then dried under avacuum at 30° C. to yield a poly-L-glutamic acid having a number averagemolecular weight (Mn) of 3600 g/mol with respect to a polyoxyethylenestandard (PEG) and an average degree of polymerization of 21.

Co-polyamino acid AB7: By a method similar to the one used for thepreparation of the co-polyamino acid AB6, applied to the hydrochloridesalt of molecule AA7 (1.43 g, 2.2 mmol) and to the co-polyamino acidAB7-1 (10.0 g), a sodium poly-L-glutamate acid modified by molecule AA7is obtained.

Dry extract: 24.3 mg/g

DP (estimated based on ¹H NMR): 21

Based on ¹H NMR: i=0.03

The calculated average molecular weight of the co-polyamino acid AB7 is3677 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn 3300 g/mol.

EXAMPLE AB8: CO-POLYAMINO ACID AB8—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA7 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3600G/MOL

Co-Polyamino Acid AB8-1:

poly-L-glutamic acid having a number average molecular weight (Mn) of3800 g/mol and a degree of polymerization of 24, originating from thepolymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated byammonia.

By a method similar to the one described in the French patent FR-A-2 801226, applied to γ-methyl-L-glutamic acid N-carboxyanhydride (25.0 g,133.6 mmol) and to a 0.5 N ammonia solution in dioxane (12.1 mL, 6.05mmol), a poly-L-glutamic acid is obtained.

Co-Polyamino Acid AB8:

By a method similar to the one used for the preparation of theco-polyamino acid AB6, applied to the hydrochloride salt of molecule AA7(2.1 g, 3.24 mmol) and to the co-polyamino acid AB8-1 (14.3 g), a sodiumpoly-L-glutamate modified by molecule AA7 is obtained.

Dry extract: 25.2 mg/g

DP (estimated based on ¹H NMR): 24

Based on ¹H NMR: i=0.03

The calculated average molecular weight of the co-polyamino acid AB8 is4099 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3600 g/mol.

EXAMPLE AB9: CO-POLYAMINO ACID AB9—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA3 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3200G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB1, applied to the hydrochloride salt of molecule AA3and to a poly-L-glutamic acid obtained by a method similar to the oneused for the preparation of the co-polyamino acid AB1-1, a sodiumpoly-L-glutamate modified by molecule AA3 is obtained.

Dry extract: 14.7 mg/g

DP (estimated based on ¹H NMR): 30

Based on ¹H NMR: i=0.12

The calculated average molecular weight of the co-polyamino acid AB9 is6192 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3200 g/mol.

EXAMPLE AB10: CO-POLYAMINO ACID AB10—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA4 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 2600G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB7, applied to the hydrochloride salt of molecule AA4and to a poly-L-glutamic acid obtained by a method similar to the oneused for the preparation of the co-polyamino acid AB1-1, a sodiumpoly-L-glutamate modified by molecule AA4 is obtained.

Dry extract: 18.3 mg/g

DP (estimated based on ¹H NMR): 25

Based on ¹H NMR: i=0.08

The calculated average molecular weight of the co-polyamino acid AB10 is4870 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=2600 g/mol.

EXAMPLE AB11: CO-POLYAMINO ACID AB11—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA5 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 2700G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB6, applied to the hydrochloride salt of molecule AA5and to a poly-L-glutamic acid obtained by a method similar to the oneused for the preparation of the co-polyamino acid AB1-1, a sodiumpoly-L-glutamate modified by molecule AA5 is obtained.

Dry extract: 20.2 mg/g

DP (estimated based on ¹H NMR): 23

Based on ¹H NMR: i=0.05

The calculated average molecular weight of the co-polyamino acid AB11 is4072 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=2700 g/mol.

EXAMPLE AB12: CO-POLYAMINO ACID AB12—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA8 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3000G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB1, applied to the hydrochloride salt of molecule AA8and to a poly-L-glutamic acid obtained by a method similar to the oneused for the preparation of the co-polyamino acid AB1-1, a sodiumpoly-L-glutamate modified by molecule AA8 is obtained.

Dry extract: 19.5 mg/g

DP (estimated based on ¹H NMR): 26

Based on ¹H NMR: i=0.04

The calculated average molecular weight of the co-polyamino acid AB12 is4477 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3000 g/mol.

EXAMPLE AB13: CO-POLYAMINO ACID AB13—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA9 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3300G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB6, applied to the hydrochloride salt of molecule AA9and to a poly-L-glutamic acid obtained by a method similar to the oneused for the preparation of the co-polyamino acid AB1-1 usingisoamylamine as initiator instead of hexylamine, a sodiumpoly-L-glutamate modified by molecule AA9 is obtained.

Dry extract: 22.3 mg/g

DP (estimated based on ¹H NMR): 35

Based on ¹H NMR: i=0.12

The calculated average molecular weight of the co-polyamino acid AB13 is7226 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3300 g/mol.

EXAMPLE AB21: CO-POLYAMINO ACID AB21—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE AA7 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3400G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB6, applied to the hydrochloride salt of molecule AA7(2.44 g, 2.4 mmol) and to a poly-L-glutamic acid (10 g) obtained by amethod similar to the one used for the preparation of the co-polyaminoacid AB1-1, a sodium poly-L-glutamate modified by molecule AA7 isobtained.

Dry extract: 22.7 mg/g

DP (estimated based on ¹H NMR): 22

Based on ¹H NMR: i=0.056

The calculated average molecular weight of the co-polyamino acid AB21 is4090 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3400 g/mol.

EXAMPLE AB24: CO-POLYAMINO ACID AB24—SODIUM POLY-L-GLUTAMATE CAPPED ATONE OF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE AA1 ANDHAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3900 G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid AB6, applied to the hydrochloride salt of molecule AA1(1.330 g, 3.08 mmol) and to a poly-L-glutamic acid having a relative Mnof 5400 g/mol (4.0 g) obtained by a method similar to the one used forthe preparation of the co-polyamino acid AB7-1, a sodiumpoly-L-glutamate capped at one of its ends by an acetyl group andmodified by molecule AA1 is obtained.

Dry extract: 18.7 mg/g

DP (estimated based on ¹H NMR): 38

Based on ¹H NMR: i=0.089

The calculated average molecular weight of the co-polyamino acid AB24 is7088 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3900 g/mol.

EXAMPLE AB25: CO-POLYAMINO ACID AB25—SODIUM POLY-L-GLUTAMATE CAPPED ATONE OF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE AA12 ANDHAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3700 G/MOL

A poly-L-glutamic acid having a relative average weight Mn of 5400 g/moland a degree of polymerization of 38 (10.0 g) obtained by a methodsimilar to the one used for the preparation of the co-polyamino acidAB7-1 is solubilized in DMF (420 mL) at 30° C., then maintained at thistemperature. In parallel, the hydrochloride salt of molecule AA12 (4.56g, 11.29 mmol) is dissolved in chloroform (60 mL), and triethylamine(1.14 g, 11.29 mmol) is added. To the solution of co-polyamino acid inDMF, NMM (7.6 g, 75.26 mmol), then HOPO (2.51 g, 22.58 mmol) are addedsuccessively. The reaction medium is then cooled to 0° C., then EDC(4.33 g, 22.58 mmol) is added, the mixture is stirred for 1 h at 0° C.,then the solution of molecule AA12 is added. The reaction medium isstirred for 2 h between 0° C. and room temperature. The reaction mediumis filtered through a woven 0.2-mm filter and poured dropwise onto 3.95L of water containing 15% by weight of NaCl and HCl (pH 2) understirring. At the end of the addition, the pH is readjusted to 2 with a37% HCl solution, and the suspension is allowed to rest overnight. Theprecipitate is collected by filtration, then solubilized in 780 mL ofwater by slow addition of a 1 N aqueous NaOH solution until the pH is 7under stirring. After filtration through a 0.45-μm filter, the solutionis diluted by addition of water to obtain a volume of 900 mL, thenacetone (485 mL) is added to obtain a solution containing 30% by weightof acetone. This solution is filtered through an activated charcoalfilter (3M R53SLP), then acetone is distilled (40° C., 100 mbar). Afterfiltration through a 0.45-μm filter, the product is purified byultrafiltration against a 0.9% aqueous NaCl solution, a carbonate buffersolution (150 mM), a 0.9% aqueous NaCl solution, a phosphate buffersolution (150 mM), a 0.9% aqueous NaCl solution, then water until theconductimetry of the permeate is less than 50 MS/cm. The solution isthen concentrated until a final volume of 600 mL is obtained. Thesolution is filtered through a 0.2-μm filter and stored at 2-8° C.

Dry extract: 19.7 mg/g

DP (estimated based on ¹H NMR): 38

Based on ¹H NMR: i=0.16

The calculated average molecular weight of the co-polyamino acid AB25 is7877 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3700 g/mol.

EXAMPLE AB28: CO-POLYAMINO ACID AB28—SODIUM POLY-L-GLUTAMATE CAPPED ATONE OF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE AA14 ANDHAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4700 G/MOL

By a method similar to the one used for the preparation of co-polyaminoacid AB6, applied to molecule AA14 (1.51 g, 2.46 mmol) and to apoly-L-glutamic acid having a relative Mn of 5400 g/mol (3.27 g)obtained by a method similar to the one used for the preparation of theco-polyamino acid AB7-1, a sodium poly-L-glutamate capped at one of itsends by an acetyl group modified by molecule AA14 is obtained afterpurification by ultrafiltration against a 0.9% aqueous NaCl solution, acarbonate buffer solution (150 mM), a 0.9% aqueous NaCl solution, aphosphate buffer solution (150 mM), 0.9% aqueous NaCl solution, thenwater until the conductimetry of the permeate is less than 50 μS/cm. Thesolution of co-polyamino acid is then concentrated to approximately 20g/L theoretical and the pH is adjusted to 7. The aqueous solution isfiltered through a 0.2-μm filter and stored at 4° C.

Dry extract: 6.1 mg/g

DP (estimated based on ¹H NMR): 38

Based on ¹H NMR: i=0.1

The calculated average molecular weight of the co-polyamino acid AB28 is8062 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4700 g/mol.

Part B:

BB: Synthesis of the Hydrophobic Molecules in which p=2

The radicals are presented in the following table by the correspondinghydrophobic molecule before grafting onto the co-polyamino acid.

TABLE 1d list of the hydrophobic moleculaes synthesized according to theinvention. Structure of the hydrophobic molecule before grafting ontothe co-polyamino No. acid BA1

BA2

BA3

BA4

BA5

BA6

BA7

Part BA: Synthesis of the Hydrophobic Molecules in which p=2

EXAMPLE BA1: MOLECULE BA1 Molecule B1: Product Obtained by the ReactionBetween Decanoic Acid and L-proline.

Dicyclohexyl carbodiimide (DCC) (16.29 g, 78.96 mmol) andN-hydroxysuccinimide (NHS) (9.09 g, 78.96 mmol) are successively addedto a solution of decanoic acid (14.28 g, 82.91 mmol) in THF (520 mL) at0° C. After 60 h of stirring at room temperature, the mixture is cooledto 0° C. for 20 min, filtered through a sintered filter. L-Proline (10g, 86.86 mmol), diisopropylethylamine (DIPEA) (68.8 mL) and water (60mL) are added to the filtrate. After 24 h of stirring at roomtemperature, the medium is diluted with water (300 mL). The aqueousphase is washed with ethyl acetate (2×250 mL), acidified to pH˜1 with a1 N aqueous HCl solution, then extracted with dichloromethane (3×150mL). The combined organic phases are dried over Na₂SO₄, filtered,concentrated under a vacuum, and the residue is purified bychromatography on silica gel (cyclohexane, ethyl acetate).

Yield: 14.6 g (69%)

¹H NMR (CDCl₃, ppm): 0.87 (3H); 1.26 (12H); 1.65 (2H); 2.02 (3H); 2.34(2H); 2.41 (1H); 3.48 (1H); 3.56 (1H); 4.58 (1H).

LC/MS (ESI): 270.2; (calculated ([M+H]⁺): 270.4)

Molecule B2: Product Obtained by the Reaction Between Molecule B1 andL-lysine.

By a method similar to the one used for the preparation of molecule B1,applied to molecule B1 (14.57 g, 54.07 mmol) and to L-lysine (4.15 g,28.39 mmol), a yellow oil is obtained.

Yield: 16.4 g (93%)

¹H NMR (CDCl₃, ppm): 0.88 (6H); 1.26 (24H); 1.35-1.65 (8H); 1.85-2.35(12H); 2.53 (0.2H); 2.90 (0.8H); 3.45-3.75 (5H); 4.50-4.70 (3H); 7.82(1H).

LC/MS (ESI): 649.6; (calculated ([M+H]⁺): 649.9).

Molecule B3: Product Obtained by Reaction Between Molecule B2 andBoc-ethylenediamine.

DIPEA (8.80 mL) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU, 8.52 g, 26.54 mmol) at room temperature areadded to a solution of molecule 82 (16.4 g, 25.27 mmol) in THF (170 mL).After 30 min of stirring. Boc-ethylenediamine (4.45 g, 27.8 mmol) isadded. After stirring at room temperature for 2 h, the solvent isevaporated at reduced pressure and the residue is diluted with ethylacetate (400 mL). The organic phase is washed with water (250 mL), asaturated aqueous NaHCO₃ solution (250 mL), a 1 N aqueous HCl solution(250 mL), a saturated aqueous NaCl solution (250 mL) and dried overNa₂SO₄. After filtration and concentration under a vacuum, the residueobtained is purified by chromatography on silica gel (ethyl acetate,methanol) to yield a colorless oil.

Yield: 12.8 g (64%)

¹H NMR (CDCl₃, ppm): 0.87 (6H); 1.25-1.60 (42H); 1.80-2.05 (4H);2.15-2.45 (9H); 3.10-3.75 (10H); 4.30 (1H); 4.50 (2H); 5.50 (0.6H); 5.89(0.2H); 6.15 (0.2H); 7.03 (1H); 7.47 (1H).

LC/MS (ESI): 791.8; (calculated ([M+H]⁺): 792.1).

Molecule BA1

4 N HCl solution in dioxane (20.2 mL) is added to a solution of moleculeB3 (12.78 g, 16.15 mmol) in dichloromethane (110 ml) at 5° C. After 20 hof stirring at 5° C., the medium is concentrated under a vacuum. Theresidue obtained is dissolved in methanol and evaporated under a vacuum,this operation being repeated 4 times to yield a white solid of moleculeBA1 in hydrochloride salt form.

Yield: 11.4 g (97%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.25-1.50 (33H); 1.57 (1H); 1.70-2.40(12H); 2.82 (2H); 3.00 (2H); 3.25-3.70 (6H); 4.05-4.50 (3H); 7.75-8.45(6H).

LC/MS (ESI): 691.6; (calculated ([M+H]⁺): 692.0).

EXAMPLE BA2: MOLECULE BA2 Molecule 4: Product Obtained by the ReactionBetween Lauric Acid and L-proline.

By a method similar to the one used for the preparation of molecule B1,applied to lauric acid (31.83 g, 157.9 mmol) and to L-proline (20 g,173.7 mmol), a yellow oil is obtained.

Yield: 34.3 g (73%)

¹H NMR (CDCl₃, ppm): 0.87 (3H); 1.26 (16H); 1.70 (2H); 1.90-2.10 (3H);2.35 (2H); 2.49 (1H); 3.48 (1H); 3.56 (1H); 4.60 (1H).

LC/MS (ESI): 298.2; (calculated ([M+H]⁺): 298.4).

Molecule B5: Product Obtained by the Reaction Between Molecule B4 andL-lysine.

By a method similar to the one used for the preparation of molecule B1,applied to molecule B4 (33.72 g, 113.36 mmol) and to L-lysine (8.70 g,59.51 mmol), a white solid is obtained.

Yield: 26.2 g (66%)

¹H NMR (CDCl₃, ppm): 0.88 (6H); 1.26 (321H); 1.35-1.65 (8H); 1.85-2.35(15H); 2.87 (1H); 3.40-3.75 (5H); 4.50-4.75 (31H); 7.87 (1H).

LC/MS (ESI): 705.6; (calculated ([M+H]⁺): 706.0).

Molecule B6: Product Obtained by Reaction Between Boc-ethylenediamineand Molecule B5.

By a method similar to the one used for the preparation of molecule B3,applied to molecule B5 (25.74 g, 36.51 mmol) and to Boc-ethylenediamine(6.43 g, 40.16 mmol), a colorless oil is obtained.

Yield: 30.9 g (quantitative)

¹H NMR (CDCl₃, ppm): 0.88 (6H); 1.35-1.65 (50H); 1.85-2.35 (13H);3.05-3.75 (10H); 4.25-4.65 (3H); 5.50 (0.4H); 5.88 (0.2H); 6.16 (0.2H);7.08 (1H); 7.26 (1H); 7.49 (0.2H)

LC/MS (ESI): 847.8; (calculated ([M+H]⁺): 848.2).

Molecule BA2

After a method similar to the one used for the preparation of moleculeBA1, applied to molecule B6 (30.9 g, 36.47 mmol), the residue obtainedafter concentration under a vacuum is dissolved in methanol andevaporated under a vacuum, this operation being repeated 4 times toyield a white solid of molecule BA2 in hydrochloride salt form afterdrying at reduced pressure.

Yield: 27.65 g (97%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.10-2.40 (54H); 2.75-3.15 (4H);3.25-3.60 (6H); 4.05-4.50 (3H); 7.50-8.50 (6H).

LC/MS (ESI): 747.6; (calculated ([M+H]⁺): 748.1).

EXAMPLE BA3: MOLECULE BA3 Molecule B7: Product Obtained by the ReactionBetween Myristic Acid and L-proline.

By a method similar to the one used for the preparation of molecule B1,applied to myristic acid (18.93 g, 82.91 mmol) and to L-proline (10 g,86.86 mmol), a yellowish oil is obtained.

Yield: 20 g (78%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.28 (20H); 1.70 (2H); 1.90-2.10 (3H);2.36 (2H); 2.51 (1H); 3.47 (1H); 3.56 (1H); 4.61 (1H).

LC/MS (ESI): 326.2; (calculated ([M+H]⁺): 326.6).

Molecule B8: Product Obtained by the Reaction Between Molecule B7 andL-lysine

By a method similar to the one used for the preparation of molecule B1,applied to molecule B7 (20.02 g, 61.5 mmol) and to L-lysine (4.72 g,32.29 mmol), a white solid is obtained.

Yield: 12.3 g (53%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.26 (40H); 1.35-1.50 (6H); 1.50-2.10(10H); 2.10-2.25 (4H); 3.01 (2H); 3.31-3.55 (4H); 4.10-4.40 (3H); 7.68(0.6H); 7.97 (1H); 8.27 (0.4H); 12.50 (1H).

LC/MS (ESI): 761.8; (calculated ([M+1]⁺): 762.1).

Molecule B9: Product Obtained by the Reaction BetweenBoc-ethylenediamine and Molecule B8.

By a method similar to the one used for the preparation of molecule B3,applied to molecule B8 (12 g, 15.77 mmol) and to Boc-ethylenediamine(3.03 g, 18.92 mmol), a colorless oil is obtained after purification bycolumn chromatography on silica gel (ethyl acetate, methanol).

Yield: 12.5 g (88%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.20-1.55 (55H); 1.50-2.25 (14H);2.95-3.10 (6H); 3.31-3.55 (4H); 4.10-4.40 (3H); 6.74 (1H); 7.60-8.25(3H).

LC/MS (ESI): 904.1; (calculated ([M+H]⁺): 904.3).

Molecule BA3

By a method similar to the one used for the preparation of molecule BA1,applied to molecule B9 (12.5 g, 13.84 mmol), the residue obtained afterconcentration under a vacuum is dissolved in methanol and evaporatedunder a vacuum, this operation being repeated 4 times to yield a whitesolid of molecule BA3 in hydrochloride salt form after drying at reducedpressure.

Yield: 9.2 g (79%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.10-1.65 (48H); 1.70-2.35 (12H); 2.85(2H); 3.01 (2H); 3.25-3.65 (6H); 4.10-4.50 (3H); 7.70-8.40 (6H).

LC/MS (ESI): 803.9; (calculated ([M+H]⁺): 804.2).

EXAMPLE BA4: MOLECULE BA4

Molecule B10: Product Obtained by the Reaction Between Molecule B8 andBoc-1-amino-4,7,10-trioxa-3-tridecane.

By a method similar to the one used for the preparation of molecule B3,applied to molecule B8 (29.80 g, 39.15 mmol) and toBoc-1-amino-4,7,10-trioxa-13-tridecane (15.05 g, 46.96 mmol), a thickcolorless oil is obtained.

Yield: 25.3 g (61%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.25-2.35 (75H); 2.85-3.20 (6H);3.25-3.65 (16H); 4.10-4.45 (3H); 6.38 (0.1H); 6.72 (0.9H); 7.50-8.25(3H).

LC/MS (ESI): 1064.2; (calculated ([M+H]⁺: 1064.5).

Molecule BA4

After a method similar to the one used for the preparation of moleculeBA1, applied to molecule B10 (25.3 g, 23.8 mmol), the residue obtainedafter concentration under a vacuum is dissolved in methanol andevaporated under a vacuum, this operation being repeated 4 times toyield a white solid of molecule BA4 in hydrochloride salt form afterdrying at reduced pressure.

Yield: 20.02 g (84%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.15-2.35 (66H); 2.80-3.20 (6H);3.30-3.65 (16H); 4.10-4.45 (3H); 7.55-8.60 (6H).

LC/MS (ESI): 964.9; (calculated ([M+H]⁺): 964.6).

EXAMPLE BA5: MOLECULE BA5 Molecule B11: Product Obtained by ReactionBetween Palmitoyl Chloride and L-proline.

By a method similar to the one used for the preparation of molecule A26,applied to palmitoyl chloride (15.39 g, 55.99 mmol) and to L-proline(12.89 &g 111.98 mmol), a white solid of molecule B11 is obtained.

Yield: 19.10 g (96%)

¹H NMR (CDCl₃, ppm): 0.88 (3H); 1.19-1.45 (24H); 1.58-1.74 (2H);1.88-2.14 (3H); 2.15-2.54 (3H); 3.47 (1H); 3.58 (1H); 4.41 (0.1H); 4.61(0.9H) 6.60-8.60 (1H).

LC/MS (ESI): 354.4; 707.8; (calculated ([M+H]⁺): 354.3; ([2M+H]⁺):707.6).

Molecule B12: Product Obtained by Reaction Between Molecule B11 andL-lysine.

By a method similar to the one used for the preparation of molecule B1,applied to molecule B11 (19.10 g, 54.02 mmol) and to L-lysine (4.15 g,28.36 mmol), an oily residue is obtained after concentration of thereaction medium at reduced pressure. This residue is diluted in water(150 mL), washed with ethyl acetate (2×75 mL), then the aqueous phase isacidified until the pH is 1 by slow addition of 6 N HCl. The product isextracted 3 times with dichloromethane, the organic phase is dried overNa₂SO₄, then filtered and concentrated at reduced pressure to yield 11.2g of yellow oily residue. In parallel, the previous organic phase ofethyl acetate is washed with a 2 N aqueous HCl solution (2×75 mL), asaturated aqueous NaCl solution (75 mL), dried over Na₂SO₄, filtered andconcentrated to yield 10.2 g of yellow oily residue. A white solid isobtained after recrystallization of each one of these residues inacetone.

Yield: 11.83 g (54%)

¹H NMR (CDCl₃, ppm): 0.87 (6H); 1.06-2.44 (70H); 2.78-2.96 (1H);3.35-3.75 (5H); 4.28-4.43 (0.1H); 4.43-4.52 (0.2H); 4.52-4.61 (1.8H);4.61-4.75 (0.9H); 7.74-8.02 (2H).

LC/MS (ESI): 818.0; (calculated ([M+H]⁺): 818.7).

Molecule B13: Product Obtained by Coupling Between Molecule B12 andBoc-ethylenediamine

By a method similar to the one used for the preparation of molecule A27,applied to molecule B12 (18.00 g, 22.02 mmol) in solution in THF and toBoc-ethylenediamine (4.23 g, 26.43 mmol), a white solid is obtainedafter recrystallization two times in acetonitrile.

Yield: 17.5 g (83%)

¹H NMR (DMSO-d₆, ppm): 0.85 (6H); 1.15-2.29 (79H); 2.92-3.12 (6H);3.30-3.59 (4H); 4.06-4.13 (0.65H); 4.16-4.29 (2H); 4.38-4.42 (0.35H);6.71-6.76 (1H); 7.60-7.69 (1.3H); 7.76-7.81 (0.65H); 7.93-7.97 (0.35H);8.00-8.04 (0.35H); 8.10-8.17 (0.35H).

LC/MS (ESI): 960.4; (calculated ([M+H]⁺): 960.8).

Molecule BA5

By a method similar to the one used for the preparation of molecule BA1,applied to molecule B13 (24.4 g, 25.43 mmol), the residue obtained afterconcentration under a vacuum is solubilized in dichloromethane (150 mL),the organic phase is washed 2 times with a 2 M aqueous sodium hydroxidesolution (90 mL). Acetonitrile (120 mL) is added, and thedichloromethane is eliminated by concentration at reduced pressure. Themedium is then allowed to rest for 72 h, and a white solid is obtainedafter filtration and rinsing with acetonitrile, then drying at reducedpressure. This operation is repeated 4 times.

Yield: 14.28 g (65%)

¹H NMR (DMSO-d6, ppm): 0.85 (6H); 1.06-2.32 (70H); 2.53-2.63 (2H);2.89-3.61 (10H); 4.04-4.43 (3H); 7.55-7.62 (0.65H); 7.65-7.72 (0.65H);7.80 (0.65H); 7.91 (0.35H); 8.03 (0.35H); 8.14-8.23 (0.35H).

LC/MS (ESI): 860.0; (calculated ([M+H]⁺): 860.8).

EXAMPLE BA6: MOLECULE BA6

Molecule B14: Product Obtained by Coupling Between Molecule A26 and2,3-diaminopropionic acid

By a method similar to the one used for the preparation of molecule B1,applied to molecule A26 (80.00 g, 245.78 mmol) and to2,3-diaminopropionic dihydrochloride (22.84 g, 129.04 mmol), a whitesolid is obtained after recrystallization in acetonitrile.

Yield: 69 g (78%)

¹H NMR (DMSO-d6, ppm): 0.86 (6H); 1.08-1.38 (40H); 1.40-1.55 (4H);1.68-2.30 (12H); 3.16-3.66 (6H); 4.20-4.39 (3H); 7.67-8.31 (2H); 12.70(1H).

LC/MS (ESI): 719.4; 741.5; (calculated ([M+H]⁺): 719.6; ([M+Na]⁺):741.6).

Molecule B15: Product Obtained by Coupling Between Molecule B14 andBoc-ethylenediamine

By a method similar to the one used for the preparation of molecule A27,applied to molecule B14 (32.00 g, 44.50 mmol) in solution indichloromethane and to Boc-ethylenodiamine (8.56 g, 53.40 mmol), acolorless oil is obtained after purification by chromatography on silicagel (ethyl acetate, methanol).

Yield: 24.5 g (64%)

¹H NMR (DMSO-d6, ppm): 0.85 (6H); 1.16-2.42 (65H); 2.89-3.14 (4H);3.17-3.66 (6H); 4.11-4.43 (3H); 6.77 (1H); 7.38-8.23 (3H).

LC/MS (ESI): 861.7; (calculated ([M+H]⁺): 861.7).

Molecule BA6

By a method similar to the one used for the preparation of molecule BA5,applied to molecule B15 (24.50 g, 28.45 mmol), a white solid is obtainedafter recrystallization in acetonitrile.

Yield: 19.7 g (91%)

¹H NMR (DMSO-d6, ppm): 0.85 (6H); 1.10-2.40 (58H); 2.51-2.62 (2H);2.90-3.16 (2H); 3.16-3.67 (6H); 4.04-4.47 (3H); 7.33-8.27 (3H).

LC/MS (ESI): 761.5; (calculated ([M+H]⁺): 761.6).

EXAMPLE BA7: MOLECULE BA7

Molecule B16: Product Obtained by the Reaction BetweenN-(tert-butoxycarbonyl)-1,6-diaminohexane and Molecule B8

By a method similar to the one used for the preparation of molecule A27,applied to molecule B8 (10 g, 13.14 mmol) and toN-(tert-butoxycarbonyl)-1,6-diaminohexane (3.41 g, 15.77 mmol) indichloromethane, a white solid is obtained after recrystallization inacetonitrile.

Yield: 10.7 g (85%)

¹H NMR (CDCl₃, ppm): 0.88 (6H); 1.17-2.40 (79H); 3.00-3.71 (10H);4.26-4.58 (3H); 4.67 (1H); 6.74 (1H); 7.34-7.49 (2H).

LC/MS (ESI): 959.9; (calculated ([M+H]⁺): 959.8).

Molecule BA7

After a method similar to the one used for the preparation of moleculeBA1, applied to molecule B16 (10.5 g, 10.94 mmol), a 2 N aqueous NaOHsolution is added dropwise to the reaction medium cooled to 0° C. Theaqueous phase is extracted with dichloromethane, then the organic phaseis washed 3 times with a 5% aqueous NaCl solution. After drying overNa₂SO₄, the organic phase is filtered, concentrated under a vacuum andthe residue is recrystallized in acetonitrile.

Yield: 5.4 g (58%)

¹H NMR (CDCl₃, ppm): 0.88 (6H); 1.19-2.40 (72H); 2.67 (2H); 3.03-3.70(8H); 4.26-4.57 (3H); 6.71 (1H); 7.39-7.49 (2H).

LC/MS (ESI): 859.8; (calculated ([M+H]⁺): 859.7).

BB: Synthesis of the Co-Polyamino Acids Statistical Co-Polyamino Acidsof Formulas VII or VIIa

TABLE 1e No. co-polyamino acids bearing carboxylate charges andhydrophobic radicals BB1

BB2

BB3

BB4

BB5

BB6

BB7

BB8

BB9

BB10

BB11

BB12

BB13

R₁ = H or pyroglutamate

Part BB: Synthesis of the Co-Polyamino Acids EXAMPLE BB1: CO-POLYAMINOACID BB1—SODIUM POLY-L-GLUTAMATE MODIFIED BY MOLECULE BA2 AND HAVING ANUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 2400 G/MOL

Co-Polyamino Acid BB1-1:

poly-L-glutamic acid having a relative number average molecular weight(Mn) of 3860 g/mol originating from the polymerization ofγ-benzyl-L-glutamate N-carboxyanhydride initiated by hexylamine

γ-Benzyl-L-glutamate N-carboxyanhydride (90.0 g, 342 mmol) is placed for30 min under a vacuum in a round-bottom flask dried beforehand in theoven, then anhydrous DMF (465 mL) is introduced. The mixture is thenstirred under argon until the dissolution is complete, cooled to 4° C.,then hexylamine (1.8 mL, 14 mmol) is introduced rapidly. The mixture isstirred between 4° C. and room temperature for 2 days. The reactionmixture is then heated at 65° C. for 4 h, cooled to room temperature,then poured dropwise into cold diisopropyl ether (6 L) under stirring.The white precipitate is recovered by filtration, washed withdiisopropyl ether (500 mL, then 250 mL), then dried under a vacuum at30° C. to yield a poly(γ-benzyl-L-glutamic) acid (PBLG).

A 33% hydrobromic acid solution (HBr) in acetic acid (135 mL, 0.77 mol)is added dropwise to a solution of PBLG (42.1 g) in trifluoroacetic acid(TFA, 325 mL) at 4° C. The mixture is stirred at room temperature for 2h, then poured dropwise onto a 1:1 (v/v) mixture of diisopropyl etherand water under stirring (1.6 L). After 1 h 30 of stirring, theheterogeneous mixture is allowed to rest overnight. The whiteprecipitate is recovered by filtration, washed with a 1:1 (v/v) mixtureof diisopropyl ether and water (200 mL).

The solid obtained is then solubilized in water (1 L) by adjusting thepH to 7 by addition of a 10 N aqueous sodium hydroxide solution, then a1 N aqueous sodium hydroxide solution. After solubilization, thetheoretical concentration is adjusted to 25 g/L theoretical by additionof water to obtain a final volume of 1.5 L.

The solution is filtered through a 0.45-μm filter, then purified byultrafiltration against a 0.9% NaCl solution, then water until theconductimetry of the permeate is less than 50 μS/cm.

The aqueous solution is then acidified by addition of 37% hydrochloricacid solution until the pH is 2. After 4 h of stirring, the precipitateobtained is filtered, then dried under a vacuum at 30° C. to yield apoly-L-glutamic acid having a number average molecular weight (Mn) of3860 g/mol with respect to a polyoxyethylene standard (PEG).

Co-Polyamino Acid BB1

The co-polyamino acid BB-1 (10.0 g) is solubilized in DMF (700 mL) at30-40° C., then cooled to 0° C. The hydrochloride salt of molecule BA2(2.95 g, 3.8 mmol) is suspended in DMF (45 mL), and triethylamine (0.39g, 3.8 mmol) is then added to this suspension, then the mixture isheated slightly under stirring until the dissolution is complete.N-Methylmorpholine (NMM, 7.6 g, 75 mmol) in DMF (14 mL) and ethylchloroformate (ECF, 8.1 g, 75 mmol) are added to the solution ofco-polyamino acid at 0° C. After 10 min at 0° C., the solution ofmolecule BA2 is added and the medium is maintained at 30° C. for 1 h.The reaction medium is poured dropwise onto 6 L of water containing 15%by weight of sodium chloride and HCl (pH 2), then it is allowed to restovernight. The precipitate is collected by filtration, washed by thesodium chloride solution at pH 2 (1 L) and dried under a vacuum forapproximately 1 h. The white solid obtained is taken up in water (600mL) and the pH is adjusted to 7 by slow addition of a 1 N aqueous NaOHsolution. The volume is adjusted to 700 mL by addition of water. Afterfiltration through a 0.45-μm filter, the clear solution obtained ispurified by ultrafiltration against a 0.9% NaCl solution, then wateruntil the conductimetry of the permeate is less than 50 μS/cm. Afterdischarging, the solution is filtered through a 0.2-μm filter and storedat 2-8° C.

Dry extract: 19.7 mg/g

DP (estimated based on ¹H NMR): 23

Based on ¹H NMR: i=6.05

The calculated average molecular weight of the co-polyamino acid BB1 is4350 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=2400 g/mol.

EXAMPLE BB2: CO-POLYAMINO ACID BB2—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA2 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4900G/MOL

A poly-L-glutamic acid having a number average molecular weight (Mn) of4100 g/mol (5.0 g) obtained by a method similar to the one used for thepreparation of the co-polyamino acid BB1-1 is solubilized in DMF (205mL) at 30-40° C., then maintained at this temperature. In parallel, thehydrochloride salt of molecule BA2 (1.44 g, 1.84 mmol) is suspended inDMF (10 mL), and triethylamine (0.19 g, 1.84 mmol) is added, then themixture is heated slightly under stirring until the dissolution iscomplete. To the solution of co-polyamino acid in DMF, NMM (3.7 g, 36.7mmol), the solution of molecule BA2, then 2-hydroxypyridine N-oxide(HOPO, 0.31 g, 2.76 mmol) are added successively. The reaction medium isthen cooled to 0° C., then EDC (0.53 g, 2.76 mmol) is added and thetemperature of the medium is raised again to room temperature for 3 h.The reaction medium is poured dropwise onto 1.55 L of water containing15% by weight of NaCl and HCl (pH 2) under stirring. At the end of theaddition, the pH is readjusted to 2 with a 1 N HCl solution, and thesuspension is about to rest overnight. The precipitate is collected byfiltration, then rinsed with 100 mL of water. The white solid obtainedis dissolved in 200 mL of water by slow addition of a 1 N aqueous NaOHsolution until the pH is 7 under stirring, then the solution is filteredthrough a 0.45-μm filter. The clear solution obtained is purified byultrafiltration against a 0.9% NaCl solution, then water until theconductimetry of the permeate is less than 50 μS/cm. The solutionobtained is filtered through a 0.2-μm filter and stored at 2-8° C.

Dry extract: 16.3 mg/g

DP (estimated based on ¹H NMR): 21

Based on ¹H NMR: i=0.047

The calculated average molecular weight of the co-polyamino acid BB2 is3932 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4900 g/mol.

EXAMPLE BB3; CO-POLYAMINO ACID BB3—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA2 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 6400G/MOL

Co-Polyamino Acid BB3-1:

poly-L-glutamic acid having a number average molecular weight (Mn) of17500 g/mol, originating from the polymerization of γ-methyl-L-glutamateN-carboxyanhydride initiated by L-leucinamide.

A poly-L-glutamic acid having a relative number average weight (Mn) of17500 g/mol at a methyl polymethacrylate standard (PMMA) is obtained bypolymerization of the γ-methyl N-carboxyanhydride of glutamic acid usingL-leucinamide as initiator and carrying out a deprotection of the methylesters by using a 37% hydrochloric acid solution according to the methoddescribed in the patent application FR-A-2 801 226.

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA2(3.23 g, 4.1 mmol) and to the co-polyamino acid BB3-1 (11 g), a sodiumpoly-L-glutamate modified by molecule BA2 is obtained.

Dry extract: 27.5 mg/g

DP (estimated based on ¹H NMR): 34

Based on ¹H NMR: li=0.049

The calculated average molecular weight of the co-polyamino acid BB3 is6405 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=6400 g/mol.

EXAMPLE BB4: CO-POLYAMINO ACID BB4—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA2 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 10500G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA2(5 g, 6.35 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=10800 g/mol (21.7 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB1-1, asodium poly-L-glutamate modified by molecule BA2 is obtained.

Dry extract: 28.2 mg/g

DP (estimated based on ¹H NMR): 65

Based on ¹H NMR: i=0.04

The calculated average molecular weight of the co-polyamino acid BB4 is11721 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=10500 g/mol.

EXAMPLE BB5: CO-POLYAMINO ACID BB5—SODIUM POLY-L-GLUTAMATE CAPPED AT ONEOF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE BA2 AND HAVING ANUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3600 G/MOL

Co-Polyamino Acid. BB5-1:

poly-L-glutamic acid having a Mn of 3700 g/mol originating from thepolymerization of γ-benzyl-L-glutamate N-carboxyanhydride initiated byhexylamine and capped at one of its ends by an acetyl group.

γ-Benzyl-L-glutamate N-carboxyanhydride (100.0 g, 380 mmol) is placedunder a vacuum for 30 minutes in a round-bottom flask dried beforehandin the oven, then anhydrous DMF (250 mL) is introduced. The mixture isthen stirred under argon until the dissolution is complete, cooled to 4°C., then hexylamine (2.3 mL, 17 mmol) is introduced rapidly. The mixtureis stirred between 4° C. and room temperature for 2 days, thenprecipitated in diisopropyl ether (3.4 L). The precipitate is recoveredby filtration, washed two times with diisopropyl ether (225 mL), thendried to yield a white solid which is dissolved in 450 mL of THF. Tothis solution, N,N-diisopropylethylamine (DIPEA, 31 mL, 176 mmol), thenacetic anhydride (17 mL, 176 mmol) are added successively. After onenight of stirring at room temperature, the solution is poured slowlyinto diisopropyl ether (3 L) over a duration of 30 min and understirring. After 1 h of stirring, the precipitate is filtered, washed twotimes with diisopropyl ether (200 mL), then dried under a vacuum at 30°C. to yield a poly(γ-benzyl-L-glutamic) acid capped at one of its endsby an acetyl group.

A 33% hydrobromic acid solution (HBr) in acetic acid (235 mL, 1.34 mol)is added dropwise to a solution of capped co-polyamino acid (72 g) intrifluoroacetic acid (TFA, 335 mL) at 4° C. The mixture is stirred atroom temperature for 3 h 30, then poured dropwise onto a 1:1 (v/v)mixture of diisopropyl ether and water under stirring (4 L). After 2 hof stirring, the heterogeneous mixture is allowed to rest overnight. Thewhite precipitate is recovered by filtration, washed with a 1:1 (v/v)mixture of diisopropyl ether and water (340 mL) then with water (340mL). The solid obtained is then solubilized in water (1.5 L) byadjusting the pH to 7 by addition of a 10 N aqueous sodium hydroxidesolution, then a 1 N aqueous sodium hydroxide solution. Aftersolubilization, the theoretical concentration is adjusted to 20 g/Ltheoretical by addition of water to obtain a final volume of 2.1 L. Thesolution is filtered through a 0.45-μm filter, then purified byultrafiltration against a 0.9% NaCl solution, then water until theconductimetry of the permeate is less than 50 μS/cm. The solution ofco-polyamino acid is then concentrated until a final volume of 1.8 L isobtained. The aqueous solution is then acidified by addition of 37%hydrochloric acid solution until the pH is 2. After 4 h of stirring, theprecipitate obtained is filtered, washed with water (330 mL), then driedunder a vacuum at 30° C. to yield a poly-L-glutamic acid having a numberaverage molecular weight (Mn) of 3700 g/mol with respect to apolyoxyethylene standard (PEG).

Co-Polyamino Acid BB5

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA2(6.92 g, 8.8 mmol) and to the co-polyamino acid BB5-1 (30.0 g), a sodiumpoly-L-glutamate capped at one of its ends by an acetyl group andmodified by molecule BA2 is obtained.

Dry extract: 29.4 mg/g

DP (estimated based on ¹H NMR): 23

Based on ¹H NMR: i=0.042

The calculated average molecular weight of the co-polyamino acid BB5 is4302 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3600 g/mol.

EXAMPLE BB6: CO-POLYAMINO ACID BB6—SODIUM POLY-L-GLUTAMATE CAPPED AT ONEOF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE BA2 AND HAVING ANUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4100 G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA2(5.8 g, 7.4 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=3800 g/mol (25 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB5-1 usingammonia instead of hexylamine, a sodium poly-L-glutamate capped at oneof its ends by an acetyl group and modified by molecule BA2 is obtained.

Dry extract: 27.6 mg/g

DP (estimated based on ¹H NMR): 24

Based on ¹H NMR: i=0.04

The calculated average molecular weight of the co-polyamino acid BB6 is4387 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4100 g/mol.

EXAMPLE BB7: CO-POLYAMINO ACID BB7—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA2 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4200G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA2(7.07 g, 9.0 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=3600 g/mol (30.0 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB1-1, asodium poly-L-glutamate modified by molecule BA2 is obtained.

Dry extract: 28.3 mg/g

DP (estimated based on ¹H NMR): 22

Based on ¹H NMR: i=0.042

The calculated average molecular weight of the co-polyamino acid BB7 is4039 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4200 g/mol.

EXAMPLE BB8: CO-POLYAMINO ACID BB5—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA2 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 5200G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA2(0.85 g, 1.1 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=4100 g/mol (5.0 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB1-1, asodium poly-L-glutamate modified by molecule BA2 is obtained.

Dry extract: 28.6 mg/g

DP (estimated based on ¹H NMR): 21

Based on ¹H NMR: i=0.026

The calculated average molecular weight of the co-polyamino acid BB8 is3620 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=5200 g/mol.

EXAMPLE BB9: CO-POLYAMINO ACID BB9—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA3 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4700G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA3(3.05 g, 3.6 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=4100 g/mol (10.0 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB1-1, asodium poly-L-glutamate modified by molecule BA3 is obtained.

Dry extract: 28.6 mg/g

DP (estimated based on ¹H NMR): 26

Based on ¹H NMR: i=0.05

The calculated average molecular weight of the co-polyamino acid BB9 is4982 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4700 g/mol.

EXAMPLE BB10: CO-POLYAMINO ACID BB10—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA3 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 4200G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA3(1.90 g, 2.3 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=3500 g/mol (10.0 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB1-1, asodium poly-L-glutamate modified by molecule BA3 is obtained.

Dry extract: 25.9 mg/g

DP (estimated based on ¹H NMR): 22

Based on ¹H NMR: i=0.029

The calculated average molecular weight of the co-polyamino acid BB10 is3872 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=4200 g/mol.

EXAMPLE BB11: CO-POLYAMINO ACID BB11—SODIUM POLY-L-GLUTAMATE CAPPED ATONE OF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE BA4 ANDHAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3900 G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA4(2.21 g, 2.2 mmol) and to a poly-L-glutamic acid having a number averageweight Mn=3700 g/mol (10 g) obtained by a method similar to the one usedfor the preparation of the co-polyamino acid BB5-1, a sodiumpoly-L-glutamate capped at one of its ends by an acetyl group andmodified by molecule BA4 is obtained.

Dry extract: 28.1 mg/g

DP (estimated based on ¹H NMR): 22

Based on ¹H NMR: i=0.032

The calculated average molecular weight of the co-polyamino acid BB11 is4118 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3900 g/mol.

EXAMPLE BB12: CO-POLYAMINO ACID BB12—SODIUM POLY-L-GLUTAMATE CAPPED ATONE OF ITS ENDS BY AN ACETYL GROUP AND MODIFIED BY MOLECULE BA3 ANDHAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 3900 G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB2, applied to the hydrochloride salt of molecule BA3(1.9 g, 2.3 mmol) and to a poly-L-glutamic acid having a number averageweight Mn=3600 g/mol (10 g) obtained by a method similar to the one usedfor the preparation of co-polyamino acid BB5-1, a sodiumpoly-L-glutamate capped at one of its ends by an acetyl group andmodified by molecule BA3 is obtained.

Dry extract: 26.7 mg/g

DP (estimated based on ¹H NMR): 23

Based on ¹H NMR: i=0.03

The calculated average molecular weight of the co-polyamino acid BB12 is4145 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=3900 g/mol.

EXAMPLE BB13: CO-POLYAMINO ACID BB13—SODIUM POLY-L-GLUTAMATE MODIFIED BYMOLECULE BA1 AND HAVING A NUMBER AVERAGE MOLECULAR WEIGHT (MN) OF 2800G/MOL

By a method similar to the one used for the preparation of theco-polyamino acid BB1, applied to the hydrochloride salt of molecule BA1(3.65 g, 5 mmol) and to a poly-L-glutamic acid having a number averagemolecular weight Mn=3600 g/mol (10 g) obtained by a method similar tothe one used for the preparation of the co-polyamino acid BB1-1, asodium poly-L-glutamate modified by molecule BA1 is obtained.

Dry extract: 25.6 mg/g

DP (estimated based on ¹H NMR): 25

Based on ¹H NMR: i=0.08

The calculated average molecular weight of the co-polyamino acid BB13 is5253 g/mol.

HPLC aqueous-SEC (calibrant PEG): Mn=2800 g/mol.

Part C:

The glucagon used is human glucagon originating from a peptide synthesisprocess. It originates from the company Bachem (reference 407473).

EXAMPLE C1: SOLUTION OF GLUCAGON AT 2 MG/ML

Powdered glucagon (80 mg) is introduced into a 45-mL flask. A 0.003 Naqueous hydrochloric acid solution (40 mL) is added. The glucagon powderis mixed by repeated inversions of the tube until the dissolution of theglucagon is complete. The solution of glucagon at 2 mg/mL is thenfiltered through a membrane (0.22 μm).

EXAMPLE C2: SOLUTION OF GLUCAGON AT 4 MG/ML

Powdered glucagon (160 mg) is introduced into a 45-mL flask. A 0.006 Naqueous hydrochloric acid solution (40 mL) is added. The glucagon powderis mixed by repeated inversions of the tube until the dissolution of theglucagon is complete. The solution of glucagon at 4 mg/mL is thenfiltered through a membrane (0.22 μm).

EXAMPLE C3: SOLUTION OF GLUCAGON AT 6 MG/ML

Powdered glucagon (240 mg) is introduced into a 45-mL flask. A 0.01 Naqueous hydrochloric acid solution (40 mL) is added. The glucagon powderis mixed by repeated inversions of the tube until the dissolution of theglucagon is complete. The solution of glucagon at 6 mg/mL is thenfiltered through a membrane (0.22 μm).

EXAMPLE C4: SOLUTION OF GLUCAGON AT 10 MG/ML

Powdered glucagon (400 mg) is introduced into a 45-mL flask. A 0.01 Naqueous hydrochloric acid solution (40 mL) is added. The glucagon powderis mixed by repeated inversions of the tube until the dissolution of theglucagon is complete. The solution of glucagon at 10 mg/mL is thenfiltered through a membrane (0.22 μm).

Tests were carried out to verify whether the polyamino acids make itpossible to solubilize the glucagon, and the minimum concentration ofco-polyamino acid necessary to solubilize the glucagon was determined.

EXAMPLE CA2: COMPOSITIONS OF CO-POLYAMINO ACID AB21 AT VARIABLECONCENTRATIONS AND GLUCAGON CONCENTRATION OF 1 MG/ML

X′ mg of co-polyamino acid AB21 were weighed on a precision scale, and 2mL of a 10 mM phosphate buffer solution comprising m-cresol (46 mM),glycerol (548 mM) were added. The composition is stirred untildissolution of the co-polyamino acid, then the solution is filteredthrough a membrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed at 2 mLof the solution of co-polyamino acid as prepared above, leading to acomposition comprising X mg/mL of co-polyamino acid and 1 mg/mL ofglucagon.

A visual inspection was carried out to determine whether or not a clearsolution was obtained. The result of the minimum concentration ispresented in Table 5.

EXAMPLE CA4: COMPOSITION OF CO-POLYAMINO ACID BB2 AT VARIABLECONCENTRATIONS AND A GLUCAGON CONCENTRATION OF 1 MG/ML

In the same manner as described in Example CA2, compositions comprisingX mg/mL of co-polyamino acid BB2 and 1 mg/mL of glucagon are prepared.

A visual inspection was carried out to determine whether or not a clearsolution was obtained. The result of the minimum concentration ispresented in Table 5.

EXAMPLE CA6: COMPOSITION OF CO-POLYAMINO ACID BB9 AT VARIABLECONCENTRATIONS AND A GLUCAGON CONCENTRATION OF 1 MG/ML

In the same manner as described in Example CA2, compositions comprisingX mg/mL of co-polyamino acids BB9 and 1 mg/mL of glucagon are prepared.

A visual inspection was carried out to determine whether or not a clearsolution was obtained. The result of the minimum concentration ispresented in Table 5.

TABLE 5 Minimum concentration of co-polyamino acid (in mg/mL) for thesolubilization of human glucagon (1 mg/mL). Minimum concentration (xmg/mL) of co-polyamino acid (in mg/mL) for the solubilization ExampleCo-polyamino acid of human glucagon (1 mg/mL) CA2 AB21 ≦1.25 CA4 BB2≦0.82 CA6 BB9 ≦1.25

Concentration series were prepared with co-polyamino acids according tothe invention, leading to the obtention of the following stablesolutions.

EXAMPLE CB2: SOLUTION OF CO-POLYAMINO ACID AB1 AT 15 MG/ML AND OFGLUCAGON AT 1 MG/ML

60 mg of co-polyamino acid AB1 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid AB1 as prepared above. Thenthree samples of 1 mL each of this solution are prepared and placedunder static conditions at 37° C.

EXAMPLE CB3: SOLUTION OF CO-POLYAMINO ACID AB5 AT 10 MG/ML AND OFGLUCAGON AT 1 MG/ML

40 mg of co-polyamino acid AB5 are weighed on a precision scale, and 2mL of a 10 mM phosphate buffer solution comprising m-cresol (46 mM),glycerol (548 mM) are added. The composition is stirred untildissolution of the co-polyamino acid, then the solution is filteredthrough a membrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB4: SOLUTION OF CO-POLYAMINO ACID AB7 AT 8.6 MG/ML AND OFGLUCAGON AT 1 MG/ML

34.4 mg of co-polyamino acid AB7 are weighed on a precision scale, and 2mL of a 10 mM phosphate buffer solution comprising m-cresol (46 mM),glycerol (548 mM) are added. The composition is stirred untildissolution of the co-polyamino acid, then the solution is filteredthrough a membrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB8: SOLUTION OF CO-POLYAMINO ACID BB2 AT 8 MG/ML AND OFGLUCAGON AT 1 MG/ML

32 mg of co-polyamino acid BB2 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB9: SOLUTION OF CO-POLYAMINO ACID BB5 AT 9 MG/ML AND OFGLUCAGON AT 1 MG/ML

36 mg of co-polyamino acid BB5 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB10: SOLUTION OF CO-POLYAMINO ACID BB7 AT 15.4 MG/ML AND OFGLUCAGON AT 1 MG/ML

61.6 mg of co-polyamino acid BB7 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 rpm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB11: SOLUTION OF CO-POLYAMINO ACID BB AT 7.6 MG/ML AND OFGLUCAGON AT 1 MG/ML

30.4 mg of co-polyamino acid BB8 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C are mixed with 2 mLof the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB12: SOLUTION OF CO-POLYAMINO ACID BB9 AT 4.3 MG/ML AND OFGLUCAGON AT 1 MG/ML

17.2 mg of co-polyamino acid BB9 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB13: SOLUTION OF CO-POLYAMINO ACID BB11 AT 5.9 MG/ML AND OFGLUCAGON AT 1 mg/mL

23.6 mg of co-polyamino acid BB11 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB14: SOLUTION OF CO-POLYAMINO ACID BB11 AT 8.6 MG/ML AND OFGLUCAGON AT 1 MG/ML

34.4 mg of co-polyamino acid BB11 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

EXAMPLE CB25: SOLUTION OF CO-POLYAMINO ACID AB21 AT 8.6 MG/ML AND OFGLUCAGON AT 1 MG/ML

34.4 mg of co-polyamino acid AB21 are weighed on a precision scale andadded to 2 mL of a 10 mM phosphate buffer solution comprising m-cresol(46 mM), glycerol (548 mM). The composition is stirred until dissolutionof the co-polyamino acid, then the solution is filtered through amembrane (0.22 μm).

2 mL of a glucagon solution as prepared in Example C1 are mixed with 2mL of the solution of co-polyamino acid as prepared above. Then threesamples of 1 mL each of this solution are prepared and placed understatic conditions at 37° C.

Part C′—Counter-Example Formulations

The cetyltrimethylammonium bromide (CTAB) originates from the companySigma-Aldrich (ref A6909).

The dodecyl maltoside (DDM) originates from the company Sigma-Aldrich(ref D4641).

The name (mPEG-DSPE 2000) originates from the company Interchim (refKVS081).

The myristoyl lysophosphatidyl choline (LMPC) originates from thecompany Combi-Block (ref: QE-2488).

In a manner similar to the composition described in Example CA2,compositions CEC1 to CEC10 are also prepared in a manner so as to obtainthe desired concentrations (Table 6d).

TABLE 6d compositions of glucagon at 1 mg/mL at pH 7.2 in the presenceof commercial products at different concentrations. Products con-Phosphate Glucagon of the centration buffer Compositions (mg/mL) priorart (mg/mL) (mM) pH CEC1 1 CTAB 0.3 2 7.13 CEC2 1 CTAB 15.7 2 7.14 CEC31 CTAB 39.3 2 7.16 CEC4 1 DDM 0.4 2 ND CEC5 1 DDM 3.2 2 7.15 CEC6 1 DDM10.7 2 7.34 CEC7 1 PEG-DSPE 2.4 2 7.20 CEC8 1 PEG-DSPE 10.9 2 7.12 CEC91 LMPC 0.4 2 ND CEC10 1 LMPC 2.1 2 7.13

Part D: Stability EXAMPLE D1: PHYSICAL STABILITY OF COMPOSITIONS OFCO-POLYAMINO ACID/GLUCAGON

The visual inspection of the samples placed under static conditions at37° C. is carried out at 0, 7, 14 and 21 days at 37° C. in order todetect the appearance of visible particles or turbidity. This inspectionis carried out according to the recommendations of the EuropeanPharmacopoeia (EP 2.9.20): the samples are subjected to illumination ofat least 2000 lux and are observed on a white background and a blackbackground. When particles are visible in at least 2 of the 3 samples,the composition is considered unstable. Thus, stable means that on theday of inspection at least 2 samples were free of particles.

The results of the visual inspections are reported in the followingTable 7.

The study of the physical stabilities of the compositions of ExamplesCB2 to CEC9 described in the table below was carried out on volumes of 1mL of composition in flasks having a capacity of 3 mL (Adelphi—ref:VCDIN2RDLS1).

TABLE 7 Results of the visual inspections of compositions comprising aco-polyamino acid and glucagon Co-Polyamino acid and product StableStable Stable of the prior Glucagon Exenatide L-methionine at 7 at 14 at21 Example art (mg/mL) (mg/mL) (mg/mL) (mg/mL) days days days CB2 AB1(15) 1 0 0 yes no no CB3 AB5 (10) 1 0 0 yes yes yes CB4 AB7 (8.6) 1 0 0yes yes yes CB8 BB2 (8) 1 0 0 yes yes yes CB9 BB5 (9) 1 0 0 yes yes noCB10 BB7 (15.4) 1 0 0 yes yes yes CB11 BB8 (7.6) 1 0 0 yes no no CB12BB9 (4.3) 1 0 0 ves yes yes CB13 BB11 (5.9) 1 0 0 ves yes no CB14 BB11(8.6) 1 0 0 yes yes yes CB25 AB21 (8.6) 1 0 0 yes yes yes CB26 AB24(6.1) 1 0 0 yes yes yes CB27 AB24 (3.0) 1 0 0 yes yes yes CB30 AB25(6.7) 1 0 0 yes yes yes CB31 AB28(6.9) 1 0 0 yes yes — CEC1 CTAB (0.3) 10 0 No — — CEC6 DDM (10.7) 1 0 0 No — — CEC7 mPEG-DSPE (2.4) 1 0 0 No —— CEC9 LMPC (0.4) 1 0 0 No — — CEC10 LPMC (2.1) 1 0 0 No — — “—” meansnot observed

EXAMPLE D2: CHEMICAL STABILITY OF COMPOSITIONS OF CO-POLYAMINOACID/GLUCAGON

An RP-HPLC method adapted based on the USP instructions was used todetermine the concentration of glucagon and of its degradation products.This method was used in order to evaluate the chemical stability of theglucagon of the compositions. The HPLC conditions are as follows:

-   -   Column: 4.6×150 mm, C-18    -   Mobile phase A: Solution S/acetonitrile 80/20 (v/v), solution S        being a 150 mM potassium dihydrogenophosphate solution in water,        adjusted to pH 2.7 with an 85% phosphoric acid solution    -   Mobile phase B: water/acetonitrile 60/40 (v/v)    -   Mobile phase C: water/acetonitrile 10/90 (v/v)    -   Column temperature: 45° C.    -   Detection: UV 210 nm    -   Temperature of the autosampler: 4° C.

The recovery was measured on samples at 7, 14 and 21 days at 37° C.under static conditions. The chemical stability data, that is to say theglucagon recovery obtained by RP-HPLC, is presented in the followingTable 8.

The study of the chemical stabilities of the compositions described inthe table below was carried out on compositions in flasks (1 mL ofcomposition in flask having a capacity of 3 mL (Adelphi—ref:VCDIN2RDLS1)).

TABLE 8 Measurements of recovery of compositions comprising aco-polyamino acid and glucagon Recovery Recovery Recovery Co-PolyaminoGlucagon Exenatide L-methionine at 7 at 14 at 21 Example acid (mg/mL)(mg/mL) (mg/mL) (mg/mL) days days days CB4 AB7 (8.6) 1 0 0 ≧95 ≧90 ≧85CB8 BB2 (8) 1 0 0 — ≧90 ≧90 CB10 BB7 (15.4) 1 0 0 ≧95 ≧90 — CB14 BB11(8-6) 1 0 0 ≧95 ≧90 ≧90 CB25 AB21 (8.6) 1 0 0 — ≧90 ≧85 CB30 AB25 1 0 0— ≧90 — CEC2 CTAB 1 0 0 — <50 “—” means not measured

Principle

The poor stability of a peptide can lead to the formation of amyloidfibrils defined as ordered macromolecular structures. These structurescan possibly result from the formation of gel within the sample.

The test of monitoring the fluorescence of Thioflavin T (ThT) is used inorder to analyze the physical stability of the solutions. Thioflavin isa small molecule probe that has a characteristic fluorescence signaturewhen it binds to fibrils of the amyloid type (Naiki et al. (1989) Anal.BioChem. 177, 244-249, LeVine (1999) Methods. Enzymol. 309, 274-284).

This method makes it possible to monitor the formation of fibrils forlow ThT concentrations within undiluted solutions. This monitoring iscarried out under accelerated stability conditions: under stirring andat 37° C.

Experimental Conditions

The samples are prepared just before the start of the measurement. Thepreparation of each composition is described in the associated example.Thioflavin T was added to the composition from a concentrated stocksolution so as to induce a negligible dilution of the composition. Theconcentration of Thioflavin T in the composition is 40 μM.

A volume of 150 μL of the composition was introduced into a well of a96-well plate, then 2.7 μL of concentrated ThT solution was introduced.Each composition was analyzed in three tests (triplicate) conducted onone plate. The plate was sealed with transparent film in order toprevent the evaporation of the composition.

This plate was then placed into the enclosure of a plate reader(EnVision 2104 Multilabel, Perkin Elmer). The temperature was regulatedat 37° C., and a lateral stirring at 960 rpm with amplitude 1 mm wascarried out.

A reading of the fluorescence intensity in each well was carried outwith an excitation wavelength of 442 nm and an emission wavelength of482 nm over time.

The fibril formation process manifests itself in a strong increase influorescence after a delay referred to as lag time.

The lag time is determined visually, taking into consideration the timewhen the fluorescence signal starts to increase significantly above thebaseline.

The lag time value reported corresponds to the average of the lag timemeasurements conducted on three wells.

The lag time results obtained are presented in the table below.

The results are presented in Table 9 below

TABLE 9 results of the latency times obtained with the compositions ofco-polyamino acid or commercial products in the presence of 1 mg/mL ofglucagon. Concentration Glucagon by weight Latency time Compositionmg/mL BC mg/mL in hours CB26 1 AB24 6.1 >68 CB30 1 AB25 6.7 >68 CB31 1AB28 6.9 >63 CEC7 1 mPEG-DSPE 2.4 2.6 CEC9 1 LMPC 0.4 3.7

The latency times obtained with the compositions of co-polyamino acid inthe presence of glucagon are higher than those of the formulations ofcommercial products in the presence of glucagon.

Compositions in the Form of an Injectable Aqueous Solution ComprisingHuman Glucagon and a Co-Polyamino Acid

The invention thus relates to physically stable compositions in the formof an injectable aqueous solution, the pH of which is from 6.0 to 8.0,comprising at least:

a) human glucagon, and

b) a co-polyamino acid bearing carboxylate charges and hydrophobicradicals Hy,

In an embodiment, the compositions according to the invention comprise,in addition, a gastrointestinal hormone.

1. A composition in the form of an injectable aqueous solution, the pHof which is from 6.0 to 8.0, comprising at least: a) human glucagon; b)a co-polyamino acid bearing carboxylate charges and hydrophobic radicalsHy, said co-polyamino acid consisting of glutamic or aspartic units, andsaid hydrophobic radicals Hy being radicals of the following formula I:

in which GpR is a radical of formula II:

GpA is a radical of formula III or III′:

GpC is a radical of formula IV:

the * indicate the sites of attachment of the different groups bound byamide functions; a is a whole number equal to 0 or 1; b is a wholenumber equal to 0 or 1; p is a whole number equal to 1 or 2, and if p isequal to 1, then a is equal to 0 or 1 and GpA is a radical of formulaIII′, and if p is equal to 2, then a is equal to 1 and GpA is a radicalof formula III; c is a whole number equal to 0 or 1, and, if c is equalto 0, then d is equal to 1 or 2; d is a whole number equal to 0, to 1 or2; r is a whole number equal to 0 or 1, and if r is equal to 0, then thehydrophobic radical of formula I is bound to the co-polyamino acid via acovalent bond between a carbonyl of the hydrophobic radical and anitrogen atom in the N-terminal position of the co-polyamino acid, thusforming an amide function originating from the reaction of an aminefunction in N-terminal position of the precursor of the co-polyaminoacid and an acid function borne by the precursor of the hydrophobicradical, and if r is equal to 1, then the hydrophobic radical of formulaI is bound to the co-polyamino acid: via a covalent bond between anitrogen atom of the hydrophobic radical and a carbonyl of theco-polyamino acid therefore forming an amide function originating fromthe reaction between an amine function of the precursor of thehydrophobic radical and an acid function borne by the precursor of theco-polyamino acid, or via a covalent bond between a carbonyl of thehydrophobic radical and a nitrogen atom in N-terminal position of theco-polyamino acid therefore forming an amide function originating fromthe reaction of an acid function of the precursor of the hydrophobicradical and an amine function in N terminal position borne by theprecursor of the co-polyamino acid; R is a radical selected from thegroup consisting of: a linear or branched divalent alkyl radicalcomprising, if GpR is a radical of formula II, from 2 to 12 carbonatoms; a linear or branched divalent alkyl radical comprising, if GpR isa radical of formula II, from 2 to 11 carbon atoms, said alkyl radicalbearing one or more —CONH2 functions, and an unsubstituted ether orpolyether radical comprising from 4 to 14 carbon atoms and from 1 to 5oxygen atoms; A is a linear or branched alkyl radical comprising from 1to 6 carbon atoms; B is a linear or branched alkyl radical, optionallycomprising an aromatic ring, comprising from 1 to 9 carbon atoms; C_(x)is a linear or branched monovalent alkyl radical, in which x indicatesthe number of carbon atoms, and: if p is equal to 1, x is from 11 to 25(11≦x≦25); if p is equal to 2, x is from 9 to 15 (9≦x≦15), the ratio ibetween the number of hydrophobic radicals and the number of glutamic oraspartic units being between 0<i≦0.5; when several hydrophobic radicalsare borne by a co-polyamino acid, then they are identical or different,the degree of polymerization DP in glutamic or aspartic units is from 10to 250; the free acid functions being in the form of a salt of analkaline cation selected from the group consisting of Na⁺ and K⁺.
 2. Thecomposition according to claim 1, wherein said hydrophobic radicals areselected from the hydrophobic radicals of formula I in which p=1,represented by the following formula V:

GpR, GpA, GpC, r and a have the definitions given above.
 3. Thecomposition according to claim 1, wherein said hydrophobic radicals areselected from the hydrophobic radicals of formula I in which a=1 andp=2, represented by the following formula VI:

in which GpR, GpA, GpC, r and a have the definitions given above.
 4. Thecomposition according to claim 1, wherein the co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is selected from theco-polyamino acids of the following formula VIIa:

in which, D represents, independently, either a —CH₂— group (asparticunit) or a —CH₂—CH₂— group (glutamic unit), Hy is a hydrophobic radicalselected from the hydrophobic radicals of formula I, V or VI, in whichr=1 and GpR is a radical of Formula II, X represents an H or a cationicentity selected from the group comprising the metal cations; n+mrepresents the degree of polymerization DP of the co-polyamino acid,that is to say the average number of monomer units per chain and5≦n+m≦250; R′1 is a radical selected from the group consisting of H, aC2 to C10 linear acyl group, a C4 to C10 branched acyl group, benzyl, aterminal “amino acid” unit and a pyroglutamate, R′2 is a —NR′R″ radical,R′ and R″, which are identical or different, being selected from thegroup consisting of H, the C2 to C10 linear or branched or cyclicalkyls, benzyl, and said alkyl R′ and R″ together optionally forming oneor more saturated, unsaturated and/or aromatic carbon rings and/oroptionally comprising heteroatoms, selected from the group consisting ofO, N and S.
 5. The composition according to claim 4, whereinco-polyamino acid bearing carboxylate charges and hydrophobic charges isselected from the co-polyamino acids of formulas VII in which R₁=R′₁ andR₂=R′₂ of the following formula VIIa:

in which m, n, X, D and Hy have the definitions given above, R′₁ is aradical selected from the group consisting of H, a C2 to C10 linear acylgroup, a C4 to C10 branched acyl group, benzyl, a terminal “amino acid”unit and a pyroglutamate, R′₂ is a —NR′R″ radical, R′ and R″, which areidentical or different, being selected from the group consisting of H,the C2 to C10 linear or branched or cyclic alkyls, benzyl, and saidalkyl R′ and R″ together optionally forming one or more saturated,unsaturated and/or aromatic rings and/or optionally comprisingheteroatoms, selected from the group consisting of O, N and S.
 6. Thecomposition according to claim 4, wherein the co-polyamino acid bearingcarboxylate charges and hydrophobic radicals is selected from theco-polyamino acids of formula VII or VIIa in which the at least oneco-polyamino acid is selected from the co-polyamino acids in which groupD is a —CH₂— group (aspartic unit).
 7. The composition according toclaim 4, wherein the co-polyamino acid bearing carboxylate charges andhydrophobic radicals is selected from the co-polyamino acids of formulasVII or VIIa in which the at least one co-polyamino acid is selected fromthe co-polyamino acids in which group D is a group —CH₂—CH₂— (glutamicunit).
 8. The composition according to claim 1, wherein theconcentration of co-polyamino acid bearing carboxylate charges andhydrophobic radicals is at most 40 mg/mL.
 9. The composition accordingto claim 1, wherein the concentration of human glucagon is from 0.25 to5 mg/mL.
 10. The composition according to claim 1, wherein the molarratio [hydrophobic radical]/[human glucagon] is less than
 15. 11. Thecomposition according to claim 1, wherein it comprises, in addition, apolyanionic compound.
 12. The composition according to claim 1, whereinit comprises, in addition, a zinc salt.
 13. The composition according toclaim 1, wherein it comprises, in addition, a gastrointestinal hormone.14. The composition according to claim 13, wherein the gastrointestinalhormone is selected from the group consisting of exenatide, liraglutide,lixisenatide, albiglutide and dulaglutide, their analogs or derivativesand their pharmaceutically acceptable salts thereof.
 15. The compositionaccording to claim 13, wherein the concentration of gastrointestinalhormone is within an interval from 0.01 to 10 mg/mL.